Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing...

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Supplementary Results Deep sequencing analyses of on-target cleavages at a clonal level In order to analyse the indel patterns at the on-target site and examine the timing of the cleavages, we carried out deep sequencing analyses of all 20 colonies shown in Figure 1e. Out of the twenty colonies analysed, seventeen showed consistent results with the flow cytometry data (Fig. 1e); nearly 100% of reads at the target site of these colonies contain indels (Supplementary Table 1). The discrepancy between the two sets of data for the remaining three colonies (Cas9#5-3, 8-3 and 8-9) can be explained by the presence of GPI-positive wild-type cells in each of the three colonies. Expansion of these cells can occur between FACS analysis and DNA extraction, as shown in a competition assay between wild type and Piga-deficient cells (Supplementary Fig. 6), and will directly result in higher number of reads derived from the wild-type sequence. It is also evident from the indel patterns shown in Supplementary Figure 5 that eighteen of the twenty colonies analysed contain single clonal indels. Since the Piga gene is X-linked, this indicates that the cleavage event occurred in the clonal origin of each colony. In particular, the Cas9#5-7 clone has a clonal 6-bp in- frame deletion (Supplementary Fig. 5), which explains the low percentage of FLAER-negative cells observed in Figure 1e. The remaining two colonies, Cas9#8-3 and Cas9#8-9, carried multiple deletions (Supplementary Fig. 5), indicating that the on-target cleavage occurred at a later stage during colony expansion. Deep sequencing analyses of off-target cleavages in cell lines constitutively expressing both Cas9 and gRNA In addition, we analysed the frequency of off-target cleavages at 275 potential off-target sites. Out of these, 95 sites have up to 5 mismatches between the genome and the Piga Site 2 gRNA and are followed by the NGG PAM. Another 95 sites have up to 5 mismatches but these contain the NAG PAM rather than the NGG PAM. The remaining 85 sites have mismatches and bulges between the sites and the gRNA and are followed by either the NGG or the NAG PAMs (Supplementary Table 1, 2). Out of the 275 potential off-target sites analysed, we identified only 2 loci (l20_5tm-2 and l20_5tm- 21) that were cleaved, albeit at different frequencies (Supplementary Table 1, 2). Site l20_5tm-21, which contains 3 mismatches, was targeted by the gRNA as efficiently as the on-target site (Supplementary Fig. 7). However, site l20_5tm-2, with 2 mismatches, displayed different cleavage frequencies in each of the twenty colonies analysed (Supplementary Table 1). Since these cell lines constitutively express both Cas9 and the gRNA, cleavages have occurred at various time points during cell expansion, which explains the various cutting frequencies and indel patterns (Supplementary Fig. 8). It is thus evident that site l20_5tm-2 represents a weak off-target site in comparison to a strong off-target site like site l20_5tm-21. Importantly, we examined 19 potential off-target sites in protein-coding regions (CCDS) and found that none showed any off-target cleavages (Supplementary Table 1, 2). Thus far, all published off-target cleavage analyses have been performed using transiently transfected samples 1-4 . To compare off-target cleavages between transient and constitutive expression of the CRISPR-Cas system, we analysed Cas9-expressing ESCs transiently transfected with the Piga Site 2 gRNA expression vector by deep sequencing. The same 2 off-target sites, which were identified in the clones constitutively expressing Cas9 and the gRNA, Nature Biotechnology: doi:10.1038/nbt.2800

Transcript of Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing...

Page 1: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Supplementary Results

Deep sequencing analyses of on-target cleavages at a clonal level

In order to analyse the indel patterns at the on-target site and examine the timing of the cleavages

we carried out deep sequencing analyses of all 20 colonies shown in Figure 1e Out of the twenty

colonies analysed seventeen showed consistent results with the flow cytometry data (Fig 1e)

nearly 100 of reads at the target site of these colonies contain indels (Supplementary Table 1) The

discrepancy between the two sets of data for the remaining three colonies (Cas95-3 8-3 and 8-9)

can be explained by the presence of GPI-positive wild-type cells in each of the three colonies

Expansion of these cells can occur between FACS analysis and DNA extraction as shown in a

competition assay between wild type and Piga-deficient cells (Supplementary Fig 6) and will

directly result in higher number of reads derived from the wild-type sequence It is also evident from

the indel patterns shown in Supplementary Figure 5 that eighteen of the twenty colonies analysed

contain single clonal indels Since the Piga gene is X-linked this indicates that the cleavage event

occurred in the clonal origin of each colony In particular the Cas95-7 clone has a clonal 6-bp in-

frame deletion (Supplementary Fig 5) which explains the low percentage of FLAER-negative cells

observed in Figure 1e The remaining two colonies Cas98-3 and Cas98-9 carried multiple

deletions (Supplementary Fig 5) indicating that the on-target cleavage occurred at a later stage

during colony expansion

Deep sequencing analyses of off-target cleavages in cell lines constitutively expressing both Cas9

and gRNA

In addition we analysed the frequency of off-target cleavages at 275 potential off-target sites Out

of these 95 sites have up to 5 mismatches between the genome and the Piga Site 2 gRNA and are

followed by the NGG PAM Another 95 sites have up to 5 mismatches but these contain the NAG

PAM rather than the NGG PAM The remaining 85 sites have mismatches and bulges between the

sites and the gRNA and are followed by either the NGG or the NAG PAMs (Supplementary Table 1

2)

Out of the 275 potential off-target sites analysed we identified only 2 loci (l20_5tm-2 and l20_5tm-

21) that were cleaved albeit at different frequencies (Supplementary Table 1 2) Site l20_5tm-21

which contains 3 mismatches was targeted by the gRNA as efficiently as the on-target site

(Supplementary Fig 7) However site l20_5tm-2 with 2 mismatches displayed different cleavage

frequencies in each of the twenty colonies analysed (Supplementary Table 1) Since these cell lines

constitutively express both Cas9 and the gRNA cleavages have occurred at various time points

during cell expansion which explains the various cutting frequencies and indel patterns

(Supplementary Fig 8) It is thus evident that site l20_5tm-2 represents a weak off-target site in

comparison to a strong off-target site like site l20_5tm-21 Importantly we examined 19 potential

off-target sites in protein-coding regions (CCDS) and found that none showed any off-target

cleavages (Supplementary Table 1 2) Thus far all published off-target cleavage analyses have been

performed using transiently transfected samples 1-4 To compare off-target cleavages between

transient and constitutive expression of the CRISPR-Cas system we analysed Cas9-expressing ESCs

transiently transfected with the Piga Site 2 gRNA expression vector by deep sequencing The same 2

off-target sites which were identified in the clones constitutively expressing Cas9 and the gRNA

Nature Biotechnology doi101038nbt2800

were also the only 2 identified in the transiently transfected cells (Supplementary Table 1 2)

Interestingly Site 2 of the Piga gene and the off-target site l20_5tm-21 displayed similar cleavage

frequencies whereas the weak off-target site l20_5tm-2 displayed lower cutting frequency

Taken together our results indicate that the CRISPR-Cas system does introduce DSBs at selected off-

target sites It is therefore important to carefully design gRNAs that are expected to have minimum

deleterious consequences such as exonic off-targets

Reference

1 Fu Y et al High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells Nature biotechnology 31 822-826 (2013)

2 Hsu PD et al DNA targeting specificity of RNA-guided Cas9 nucleases Nature biotechnology 31 827-832 (2013)

3 Mali P et al CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering Nature biotechnology 31 833-838 (2013)

4 Pattanayak V et al High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity Nature biotechnology 31 839-843 (2013)

Nature Biotechnology doi101038nbt2800

Supplementary Figure 1 Genes involved in the GPI biosynthesis pathway and 6TG genotoxicity a Sche-matic of GPI biosynthesis The first and second reactions occur at the cytoplasmic side of the ER but subse-quent reactions occur at the luminal side of the ER GPI-anchored proteins are transported to the cell surface via the Golgi apparatus Pgap2 is involved in fatty acid remodelling in the Golgi Aerolysin from Aeromonas hydrophila and alpha-toxin from Clostridium septicum are cytolytic pore-forming toxins and use GPI-anchored proteins as their receptors GPI-anchored proteins are essential for development as Piga knockout mice are embryonic lethal however GPI-deficient cells are viable b Schematic of 6TG metabolism and its genotoxicity Hprt converts 6TG into thio-GMP After further modification thio-dGTP is formed and incorporated into genomic DNA during replication resulting in DNA mispairing Mismatch repair (MMR) genes recognise the mispairing and induce apoptosis In contrast MMR-deficient cells are not able to recog-nise the mispairing and are therefore able to survive under 6TG treatment Hprt-deficient cells are also viable in the presence of 6TG since 6TG cannot be converted into a toxic molecule

AcPigaPigcPigpPigqPIgyDpm2

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Nature Biotechnology doi101038nbt2800

a bPB PBbpAEF1α Puro-2A-hCas9

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Supplementary Figure 2 Generation of mouse ESC lines expressing hCas9 by single-copy piggyBac-mediated transgenesis a Schematic of the piggyBac transposon vector carrying human elongation factor 1a (EF1a)-driven hCas9 fused with the puromycin resistant gene via self-cleavage peptide T2A PB piggyBac repeats bpA bovine growth hormone polyadenylation signal sequence The thick line represents the probe for Southern blot analysis b Southern blot analysis of hCas9-expressing ESC lines Genomic DNA was digested with HindIII and hybridized with the radiolabeled probe shown in a All clones carry a single piggyBac transpo-son Note that the shortest band size is 19kb Size markers are indicated on the right

kb

Nature Biotechnology doi101038nbt2800

Site4Site2

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Site 1 TCACTCCAGACCCATTTAGG AGGSite 2 GAAGAGAGCATCATGGGCCA TGGSite 3 CGGATTTGCTGATGTCAGCT CGGSite 4 CAGAGAAAGAACTGTGGGAA TGG

Piga

U6 gRNA scaffold Terminatorspacer

GGACGAAACACCGGGTCTTCGAGAAGACGTTTTAGAGCTAGACCTGCTTTGTGGCCCAGAAGCTCTTCTGCAAAATCTCGATCT

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5rsquo-CACC NNNNN NNNNN NNNNN NNNNN GT-3rsquo 3rsquo-nnnnn nnnnn nnnnn nnnnn CAAAAT-5rsquo

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Supplementary Figure 4 Generation of doubly transgenic ESC lines a Schematic of the piggyBac transpo-son vector carrying the gRNA expression cassette together with the G418 resistant gene cassette The thick line represents the probe for Southern blot analysis b Southern blot analysis of gRNA-expressing ESC clones Genomic DNA was digested with MscI and hybridized with the radiolabeled probe shown in a Note that the shortest band size is 12kb All clones analysed carry a single gRNA expression cassette

kb

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 5 Size distribution of indels at the on-target site of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions When deletions are smaller than 20 bp the actual deletion sizes are indicated Note that Cas98-4 has a 118-bp deletion Its read count is much lower than the rest because the corresponding small PCR products were depleted during size selection

Nature Biotechnology doi101038nbt2800

Supplementary Figure 6 A competition assay between wild type and Piga-deficient ESCs ab Flow cytom-etry analyses Piga-deficient ESCs were obtained by alpha-toxin selection of hCas9-expressing ESCs trans-fected with the Piga Site 2 gRNA expression vector Wild type and mutant ESCs were mix at a 11 ratio and cultured under feeder-free conditions in 4 replicates Flow cytometry analyses using FLAER were performed at every passage The Piga-deficient (FLAER-negative) ESC population was depleted over time Data are shown as mean plusmn sd (n=4)

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Supplementary Figure 7 Size distribution of indels at the off-target site (l20_5tm-21) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

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Nature Biotechnology doi101038nbt2800

Dpm1 (1)

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Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Rea

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Indel size [bp]

Nature Biotechnology doi101038nbt2800

minus40 minus20 0 20 40

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1000

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2000

Dpm1

minus40 minus20 0 20 40

0

1000

2000

3000

4000Dpm2

minus40 minus20 0 20 40

0

500

1000

1500

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2500

Dpm3

minus40 minus20 0 20 40

0

1000

2000

3000

4000

5000

6000

Gpaa1

minus40 minus20 0 20 40

Mpdu1

minus40 minus20 0 20 40

0

500

1000

1500

2000

2500

3000

3500

Pgap2

minus40 minus20 0 20 40

0

2000

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8000

10000

12000

14000

Piga

minus40 minus20 0 20 40

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Pigb

minus40 minus20 0 20 40

0

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4000

Pigc

minus40 minus20 0 20 40

0

2000

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8000

Pigf

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800

Pigg

minus40 minus20 0 20 40

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10000Pigh

minus40 minus20 0 20 40

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Pigk

minus40 minus20 0 20 40

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Pigl

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Pigm

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3500Pign

minus40 minus20 0 20 40

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Pigo

minus40 minus20 0 20 40

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1500Pigp

minus40 minus20 0 20 40

0

1000

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4000Pigq

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Pigu

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minus40 minus20 0 20 40

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8000

Pigw

minus40 minus20 0 20 40

0

1000

2000

3000

4000

5000

Pigx

minus40 minus20 0 20 40

Pigy

∆7bp

∆7bp∆7bp

∆7bp

∆7bp∆17bp

∆7bp

∆4bp ∆4bp ∆2bp

∆5bp

∆5bp

∆5bp ∆6bp ∆6bp

∆5bp

∆6bp ∆6bp ∆6bp

∆14bp

∆12bp

∆13bp ∆10bp ∆10bp∆14bp

NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

Pgap2

Pigg Pign

Pigt

Pigy

Dpm2

Piga

Dpm3

Pigb

Gpaa1

Pigc

Mpdu1

Pigf

Pigk Pigl Pigm

Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Pgap2

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Pigm

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Pigp

0-20-40 20 40 0-20-40 20 40 0-20-40 20 40 0-20-40 20 40

Pigx

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2000

0

1000

2000

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1000

0

500

1000

Indel size [bp]

Rea

d co

unts

Nature Biotechnology doi101038nbt2800

0

20

40

60

80

100

120

Dpm

1D

pm2

Dpm

3G

paa1

Mpd

u1P

gap2

Pig

aP

igb

Pig

cP

igf

Pig

gP

igh

Pig

kP

igl

Pig

mP

ign

Pig

oP

igp

Pig

qP

igs

Pig

tP

igu

Pig

vP

igw

Pig

xP

igy

Perc

enta

ge

Ins+Del

Ins

Del

0

20

40

60

80

100

120

Dpm

1D

pm2

Dpm

3G

paa1

Mpd

u1P

gap2

Pig

aP

igb

Pig

cP

igf

Pig

gP

igh

Pig

kP

igl

Pig

mP

ign

Pig

oP

igp

Pig

qP

igs

Pig

tP

igu

Pig

vP

igw

Pig

xP

igy

Perc

enta

ge

Ins+Del

Ins

Del

a

b

NA

NA

Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

NA

gRNA

Dep

letio

n ra

te re

lativ

eto

the

lent

ivira

l lib

rary

Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

gRN

A si

te 1

gRN

A si

te 1

Mlh1Msh6

TRC

N00

0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

Moc

k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 2: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

were also the only 2 identified in the transiently transfected cells (Supplementary Table 1 2)

Interestingly Site 2 of the Piga gene and the off-target site l20_5tm-21 displayed similar cleavage

frequencies whereas the weak off-target site l20_5tm-2 displayed lower cutting frequency

Taken together our results indicate that the CRISPR-Cas system does introduce DSBs at selected off-

target sites It is therefore important to carefully design gRNAs that are expected to have minimum

deleterious consequences such as exonic off-targets

Reference

1 Fu Y et al High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells Nature biotechnology 31 822-826 (2013)

2 Hsu PD et al DNA targeting specificity of RNA-guided Cas9 nucleases Nature biotechnology 31 827-832 (2013)

3 Mali P et al CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering Nature biotechnology 31 833-838 (2013)

4 Pattanayak V et al High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity Nature biotechnology 31 839-843 (2013)

Nature Biotechnology doi101038nbt2800

Supplementary Figure 1 Genes involved in the GPI biosynthesis pathway and 6TG genotoxicity a Sche-matic of GPI biosynthesis The first and second reactions occur at the cytoplasmic side of the ER but subse-quent reactions occur at the luminal side of the ER GPI-anchored proteins are transported to the cell surface via the Golgi apparatus Pgap2 is involved in fatty acid remodelling in the Golgi Aerolysin from Aeromonas hydrophila and alpha-toxin from Clostridium septicum are cytolytic pore-forming toxins and use GPI-anchored proteins as their receptors GPI-anchored proteins are essential for development as Piga knockout mice are embryonic lethal however GPI-deficient cells are viable b Schematic of 6TG metabolism and its genotoxicity Hprt converts 6TG into thio-GMP After further modification thio-dGTP is formed and incorporated into genomic DNA during replication resulting in DNA mispairing Mismatch repair (MMR) genes recognise the mispairing and induce apoptosis In contrast MMR-deficient cells are not able to recog-nise the mispairing and are therefore able to survive under 6TG treatment Hprt-deficient cells are also viable in the presence of 6TG since 6TG cannot be converted into a toxic molecule

AcPigaPigcPigpPigqPIgyDpm2

Dpm1Dpm2Dpm3

Mpdu1

Pigl

Pigw

PigmPigx Pigf

PigoPigfPigg

Gpaa1PigkPigsPigtPigu Pgap2Pigv Pign Pigb

Protein

Transport to cell surfaceER lumen

Cytoplasm

a

Hprt

Mlh1Msh2Msh6Pms2

N

NSH

NHH2N

N

N

NSH

NH2N

N

6-Thioguanine (6TG)

Thio-GMP Thio-dGTP

OP

N

NSH

NH2N

N

OPPP

Incorporate into genomic DNA

MMR(+) Apoptosis

MMR(-) Survive

b

Phosphatidylinositol

Dolichol phosphate

Glucosamine

Mannose

Phosphoethanolamine

Nature Biotechnology doi101038nbt2800

a bPB PBbpAEF1α Puro-2A-hCas9

HindIII

19 kb

clone 4clone 5

clone 8clone 9

clone 10clone 11

clone 12clone 3

30

506080100

20

15

10

05

Supplementary Figure 2 Generation of mouse ESC lines expressing hCas9 by single-copy piggyBac-mediated transgenesis a Schematic of the piggyBac transposon vector carrying human elongation factor 1a (EF1a)-driven hCas9 fused with the puromycin resistant gene via self-cleavage peptide T2A PB piggyBac repeats bpA bovine growth hormone polyadenylation signal sequence The thick line represents the probe for Southern blot analysis b Southern blot analysis of hCas9-expressing ESC lines Genomic DNA was digested with HindIII and hybridized with the radiolabeled probe shown in a All clones carry a single piggyBac transpo-son Note that the shortest band size is 19kb Size markers are indicated on the right

kb

Nature Biotechnology doi101038nbt2800

Site4Site2

Site3 Site1

Site 1 TCACTCCAGACCCATTTAGG AGGSite 2 GAAGAGAGCATCATGGGCCA TGGSite 3 CGGATTTGCTGATGTCAGCT CGGSite 4 CAGAGAAAGAACTGTGGGAA TGG

Piga

U6 gRNA scaffold Terminatorspacer

GGACGAAACACCGGGTCTTCGAGAAGACGTTTTAGAGCTAGACCTGCTTTGTGGCCCAGAAGCTCTTCTGCAAAATCTCGATCT

Transciption

5rsquo-CACC NNNNN NNNNN NNNNN NNNNN GT-3rsquo 3rsquo-nnnnn nnnnn nnnnn nnnnn CAAAAT-5rsquo

Duplex oligos

a

d

b

FLA

ER

-neg

ativ

e ce

lls (

)

FSC

FLA

ER

cSite 2

Site 3 No gRNA

Site 1

Supplementary Figure 3 Construction and validation of gRNA-expression vector a Schematic of the gRNA cloning vector BbsI digestion removes the spacer and produces cohesive ends which allows duplex oligonu-cleotides with compatible overhangs to ligate into Underlined BbsI sites For the duplex oligonucleotides compatible overhangs are shown in blue gRNA sequences are shown as N (top strand) and n (bottom strand) b The sequences and the genomic positions of gRNAs targeting the Piga gene The genomic PAM sequence of each gRNA is shown in red c Flow cytometry analysis of GPI-anchored protein expression Analysis was performed 6 days post transfection d A summary of the flow cytometry analyses in c Transfec-tion was performed in triplicates Data are shown as mean plusmn sd (n=3)

Site 4

0

5

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Site 1 Site 2 Site 3 Site 4 No gRNA

Nature Biotechnology doi101038nbt2800

a bU6 20-bp

guide seqgRNA scaffold T7PB PBPGKneopA

MscI

12 kb

30

506080100

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05

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

JM8-Cas5 JM8-Cas8

Supplementary Figure 4 Generation of doubly transgenic ESC lines a Schematic of the piggyBac transpo-son vector carrying the gRNA expression cassette together with the G418 resistant gene cassette The thick line represents the probe for Southern blot analysis b Southern blot analysis of gRNA-expressing ESC clones Genomic DNA was digested with MscI and hybridized with the radiolabeled probe shown in a Note that the shortest band size is 12kb All clones analysed carry a single gRNA expression cassette

kb

Nature Biotechnology doi101038nbt2800

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Cas98minus3

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-30

-118-33

Cou

nts

Indel size [bp]

Supplementary Figure 5 Size distribution of indels at the on-target site of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions When deletions are smaller than 20 bp the actual deletion sizes are indicated Note that Cas98-4 has a 118-bp deletion Its read count is much lower than the rest because the corresponding small PCR products were depleted during size selection

Nature Biotechnology doi101038nbt2800

Supplementary Figure 6 A competition assay between wild type and Piga-deficient ESCs ab Flow cytom-etry analyses Piga-deficient ESCs were obtained by alpha-toxin selection of hCas9-expressing ESCs trans-fected with the Piga Site 2 gRNA expression vector Wild type and mutant ESCs were mix at a 11 ratio and cultured under feeder-free conditions in 4 replicates Flow cytometry analyses using FLAER were performed at every passage The Piga-deficient (FLAER-negative) ESC population was depleted over time Data are shown as mean plusmn sd (n=4)

a P1 P2 P3 P4 P5 P6

b

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P1 P2 P3 P4 P5 P6

FLA

ER

-neg

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)

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Nature Biotechnology doi101038nbt2800

JM8

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Cas95minus9

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Cas98minus10

0

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nts

Indel size [bp]

Supplementary Figure 7 Size distribution of indels at the off-target site (l20_5tm-21) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

JM8

0

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Cas95minus1

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Cas95minus2

0

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Cas95minus3

0

500

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leminus20

minus15

minus10 minus5 0 5 10 15

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Cas95minus4

0

500

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leminus20

minus15

minus10 minus5 0 5 10 15

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Cas95minus5

0

500

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leminus20

minus15

minus10 minus5 0 5 10 15

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Cas95minus6

0

500

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leminus20

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minus10 minus5 0 5 10 15

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Cas95minus7

0

500

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minus10 minus5 0 5 10 15

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Cas95minus8

0

500

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leminus20

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minus10 minus5 0 5 10 15

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Cas95minus9

0

500

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minus15

minus10 minus5 0 5 10 15

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Cas95minus10

0

500

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leminus20

minus15

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Cas98minus1

0

500

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leminus20

minus15

minus10 minus5 0 5 10 15

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Cas98minus2

0

500

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Cas98minus3

0

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Cas98minus4

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Cas98minus6

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Cas98minus9

0

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Cas98minus10

0

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minus15

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Cou

nts

Indel size [bp]

Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

0

20

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100

2 8Days post infection

p=00003

Piga Site 3 endogenousPiga Site 1 endogenousPiga Site 2 endogenousPiga Site 3 alteredPiga Site 1 alteredEmpty vectorMock infection

BFP

-pos

itive

()

Nature Biotechnology doi101038nbt2800

Dpm1 (1)

Pgap2 (2)

Pigg (1)

Pign (1)

Pigt (1)

Pigy (1)

Dpm2 (1)

Piga (2)

Pigh (1)

Pigo (1)

Pigu (1)

Control

Dpm3 (1)

Pigb (1)

Pigk (1)

Pigp (1)

Pigv (2)

Gpaa1 (1)

Pigc (2)

Pigl (2)

Pigq (1)

Pigw (2)

Mpdu1 (1)

Pigf (2)

Pigm (2)

Pigs (1)

Pigx (2)

Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

BFP

Nature Biotechnology doi101038nbt2800

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Dpm1

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minus40 minus20 0 20 40

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Pigx

minus40 minus20 0 20 40

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6000Pigy

∆7bp

∆7bp

∆5bp

∆11bp∆4bp

∆2bp∆2bp ∆2bp

∆12bp

∆9bp

∆6bp

∆6bp∆9bp

∆8bp

∆5bp

∆11bp

∆18bp

∆11bp

∆6bp ∆6bp

∆6bp

∆6bp∆6bp

∆6bp

∆6bp∆15bp

∆15bp

∆5bp∆10bp

Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Rea

d co

unts

Indel size [bp]

Nature Biotechnology doi101038nbt2800

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Dpm1

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0

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Dpm3

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0

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Gpaa1

minus40 minus20 0 20 40

Mpdu1

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Pigy

∆7bp

∆7bp∆7bp

∆7bp

∆7bp∆17bp

∆7bp

∆4bp ∆4bp ∆2bp

∆5bp

∆5bp

∆5bp ∆6bp ∆6bp

∆5bp

∆6bp ∆6bp ∆6bp

∆14bp

∆12bp

∆13bp ∆10bp ∆10bp∆14bp

NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

Pgap2

Pigg Pign

Pigt

Pigy

Dpm2

Piga

Dpm3

Pigb

Gpaa1

Pigc

Mpdu1

Pigf

Pigk Pigl Pigm

Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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1D

pm2

Dpm

3G

paa1

Mpd

u1P

gap2

Pig

aP

igb

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cP

igf

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gP

igh

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kP

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

NA

gRNA

Dep

letio

n ra

te re

lativ

eto

the

lent

ivira

l lib

rary

Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

gRN

A si

te 1

gRN

A si

te 1

Mlh1Msh6

TRC

N00

0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

Moc

k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 3: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Supplementary Figure 1 Genes involved in the GPI biosynthesis pathway and 6TG genotoxicity a Sche-matic of GPI biosynthesis The first and second reactions occur at the cytoplasmic side of the ER but subse-quent reactions occur at the luminal side of the ER GPI-anchored proteins are transported to the cell surface via the Golgi apparatus Pgap2 is involved in fatty acid remodelling in the Golgi Aerolysin from Aeromonas hydrophila and alpha-toxin from Clostridium septicum are cytolytic pore-forming toxins and use GPI-anchored proteins as their receptors GPI-anchored proteins are essential for development as Piga knockout mice are embryonic lethal however GPI-deficient cells are viable b Schematic of 6TG metabolism and its genotoxicity Hprt converts 6TG into thio-GMP After further modification thio-dGTP is formed and incorporated into genomic DNA during replication resulting in DNA mispairing Mismatch repair (MMR) genes recognise the mispairing and induce apoptosis In contrast MMR-deficient cells are not able to recog-nise the mispairing and are therefore able to survive under 6TG treatment Hprt-deficient cells are also viable in the presence of 6TG since 6TG cannot be converted into a toxic molecule

AcPigaPigcPigpPigqPIgyDpm2

Dpm1Dpm2Dpm3

Mpdu1

Pigl

Pigw

PigmPigx Pigf

PigoPigfPigg

Gpaa1PigkPigsPigtPigu Pgap2Pigv Pign Pigb

Protein

Transport to cell surfaceER lumen

Cytoplasm

a

Hprt

Mlh1Msh2Msh6Pms2

N

NSH

NHH2N

N

N

NSH

NH2N

N

6-Thioguanine (6TG)

Thio-GMP Thio-dGTP

OP

N

NSH

NH2N

N

OPPP

Incorporate into genomic DNA

MMR(+) Apoptosis

MMR(-) Survive

b

Phosphatidylinositol

Dolichol phosphate

Glucosamine

Mannose

Phosphoethanolamine

Nature Biotechnology doi101038nbt2800

a bPB PBbpAEF1α Puro-2A-hCas9

HindIII

19 kb

clone 4clone 5

clone 8clone 9

clone 10clone 11

clone 12clone 3

30

506080100

20

15

10

05

Supplementary Figure 2 Generation of mouse ESC lines expressing hCas9 by single-copy piggyBac-mediated transgenesis a Schematic of the piggyBac transposon vector carrying human elongation factor 1a (EF1a)-driven hCas9 fused with the puromycin resistant gene via self-cleavage peptide T2A PB piggyBac repeats bpA bovine growth hormone polyadenylation signal sequence The thick line represents the probe for Southern blot analysis b Southern blot analysis of hCas9-expressing ESC lines Genomic DNA was digested with HindIII and hybridized with the radiolabeled probe shown in a All clones carry a single piggyBac transpo-son Note that the shortest band size is 19kb Size markers are indicated on the right

kb

Nature Biotechnology doi101038nbt2800

Site4Site2

Site3 Site1

Site 1 TCACTCCAGACCCATTTAGG AGGSite 2 GAAGAGAGCATCATGGGCCA TGGSite 3 CGGATTTGCTGATGTCAGCT CGGSite 4 CAGAGAAAGAACTGTGGGAA TGG

Piga

U6 gRNA scaffold Terminatorspacer

GGACGAAACACCGGGTCTTCGAGAAGACGTTTTAGAGCTAGACCTGCTTTGTGGCCCAGAAGCTCTTCTGCAAAATCTCGATCT

Transciption

5rsquo-CACC NNNNN NNNNN NNNNN NNNNN GT-3rsquo 3rsquo-nnnnn nnnnn nnnnn nnnnn CAAAAT-5rsquo

Duplex oligos

a

d

b

FLA

ER

-neg

ativ

e ce

lls (

)

FSC

FLA

ER

cSite 2

Site 3 No gRNA

Site 1

Supplementary Figure 3 Construction and validation of gRNA-expression vector a Schematic of the gRNA cloning vector BbsI digestion removes the spacer and produces cohesive ends which allows duplex oligonu-cleotides with compatible overhangs to ligate into Underlined BbsI sites For the duplex oligonucleotides compatible overhangs are shown in blue gRNA sequences are shown as N (top strand) and n (bottom strand) b The sequences and the genomic positions of gRNAs targeting the Piga gene The genomic PAM sequence of each gRNA is shown in red c Flow cytometry analysis of GPI-anchored protein expression Analysis was performed 6 days post transfection d A summary of the flow cytometry analyses in c Transfec-tion was performed in triplicates Data are shown as mean plusmn sd (n=3)

Site 4

0

5

10

15

20

Site 1 Site 2 Site 3 Site 4 No gRNA

Nature Biotechnology doi101038nbt2800

a bU6 20-bp

guide seqgRNA scaffold T7PB PBPGKneopA

MscI

12 kb

30

506080100

20

15

10

05

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

JM8-Cas5 JM8-Cas8

Supplementary Figure 4 Generation of doubly transgenic ESC lines a Schematic of the piggyBac transpo-son vector carrying the gRNA expression cassette together with the G418 resistant gene cassette The thick line represents the probe for Southern blot analysis b Southern blot analysis of gRNA-expressing ESC clones Genomic DNA was digested with MscI and hybridized with the radiolabeled probe shown in a Note that the shortest band size is 12kb All clones analysed carry a single gRNA expression cassette

kb

Nature Biotechnology doi101038nbt2800

JM8

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus1

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus2

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus3

0

500

1000

1500

2000

2500

3000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus4

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus5

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus6

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus7

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus8

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus9

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus10

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus1

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus2

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus3

0

500

1000

1500

2000

2500

3000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus4

0

100

200

300

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus5

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus6

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus7

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus8

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus9

0

500

1000

1500

2000

2500

3000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus10

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

-30

-118-33

Cou

nts

Indel size [bp]

Supplementary Figure 5 Size distribution of indels at the on-target site of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions When deletions are smaller than 20 bp the actual deletion sizes are indicated Note that Cas98-4 has a 118-bp deletion Its read count is much lower than the rest because the corresponding small PCR products were depleted during size selection

Nature Biotechnology doi101038nbt2800

Supplementary Figure 6 A competition assay between wild type and Piga-deficient ESCs ab Flow cytom-etry analyses Piga-deficient ESCs were obtained by alpha-toxin selection of hCas9-expressing ESCs trans-fected with the Piga Site 2 gRNA expression vector Wild type and mutant ESCs were mix at a 11 ratio and cultured under feeder-free conditions in 4 replicates Flow cytometry analyses using FLAER were performed at every passage The Piga-deficient (FLAER-negative) ESC population was depleted over time Data are shown as mean plusmn sd (n=4)

a P1 P2 P3 P4 P5 P6

b

0

10

20

30

40

50

60

70

P1 P2 P3 P4 P5 P6

FLA

ER

-neg

ativ

e (

)

Passage

Nature Biotechnology doi101038nbt2800

JM8

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus1

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus2

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus3

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus4

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus5

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus6

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus7

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus8

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus9

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus10

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus1

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus2

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus3

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus4

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

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Supplementary Figure 7 Size distribution of indels at the off-target site (l20_5tm-21) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

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Nature Biotechnology doi101038nbt2800

Dpm1 (1)

Pgap2 (2)

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Pign (1)

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Control

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Pigp (1)

Pigv (2)

Gpaa1 (1)

Pigc (2)

Pigl (2)

Pigq (1)

Pigw (2)

Mpdu1 (1)

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Pigm (2)

Pigs (1)

Pigx (2)

Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

BFP

Nature Biotechnology doi101038nbt2800

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∆7bp

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

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Nature Biotechnology doi101038nbt2800

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NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

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Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

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Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 4: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

a bPB PBbpAEF1α Puro-2A-hCas9

HindIII

19 kb

clone 4clone 5

clone 8clone 9

clone 10clone 11

clone 12clone 3

30

506080100

20

15

10

05

Supplementary Figure 2 Generation of mouse ESC lines expressing hCas9 by single-copy piggyBac-mediated transgenesis a Schematic of the piggyBac transposon vector carrying human elongation factor 1a (EF1a)-driven hCas9 fused with the puromycin resistant gene via self-cleavage peptide T2A PB piggyBac repeats bpA bovine growth hormone polyadenylation signal sequence The thick line represents the probe for Southern blot analysis b Southern blot analysis of hCas9-expressing ESC lines Genomic DNA was digested with HindIII and hybridized with the radiolabeled probe shown in a All clones carry a single piggyBac transpo-son Note that the shortest band size is 19kb Size markers are indicated on the right

kb

Nature Biotechnology doi101038nbt2800

Site4Site2

Site3 Site1

Site 1 TCACTCCAGACCCATTTAGG AGGSite 2 GAAGAGAGCATCATGGGCCA TGGSite 3 CGGATTTGCTGATGTCAGCT CGGSite 4 CAGAGAAAGAACTGTGGGAA TGG

Piga

U6 gRNA scaffold Terminatorspacer

GGACGAAACACCGGGTCTTCGAGAAGACGTTTTAGAGCTAGACCTGCTTTGTGGCCCAGAAGCTCTTCTGCAAAATCTCGATCT

Transciption

5rsquo-CACC NNNNN NNNNN NNNNN NNNNN GT-3rsquo 3rsquo-nnnnn nnnnn nnnnn nnnnn CAAAAT-5rsquo

Duplex oligos

a

d

b

FLA

ER

-neg

ativ

e ce

lls (

)

FSC

FLA

ER

cSite 2

Site 3 No gRNA

Site 1

Supplementary Figure 3 Construction and validation of gRNA-expression vector a Schematic of the gRNA cloning vector BbsI digestion removes the spacer and produces cohesive ends which allows duplex oligonu-cleotides with compatible overhangs to ligate into Underlined BbsI sites For the duplex oligonucleotides compatible overhangs are shown in blue gRNA sequences are shown as N (top strand) and n (bottom strand) b The sequences and the genomic positions of gRNAs targeting the Piga gene The genomic PAM sequence of each gRNA is shown in red c Flow cytometry analysis of GPI-anchored protein expression Analysis was performed 6 days post transfection d A summary of the flow cytometry analyses in c Transfec-tion was performed in triplicates Data are shown as mean plusmn sd (n=3)

Site 4

0

5

10

15

20

Site 1 Site 2 Site 3 Site 4 No gRNA

Nature Biotechnology doi101038nbt2800

a bU6 20-bp

guide seqgRNA scaffold T7PB PBPGKneopA

MscI

12 kb

30

506080100

20

15

10

05

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

JM8-Cas5 JM8-Cas8

Supplementary Figure 4 Generation of doubly transgenic ESC lines a Schematic of the piggyBac transpo-son vector carrying the gRNA expression cassette together with the G418 resistant gene cassette The thick line represents the probe for Southern blot analysis b Southern blot analysis of gRNA-expressing ESC clones Genomic DNA was digested with MscI and hybridized with the radiolabeled probe shown in a Note that the shortest band size is 12kb All clones analysed carry a single gRNA expression cassette

kb

Nature Biotechnology doi101038nbt2800

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Cas95minus7

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Cas95minus8

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leminus20

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Cas95minus9

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leminus20

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ge20

Cas95minus10

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ge20

Cas98minus1

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Cas98minus2

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Cas98minus3

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Cas98minus4

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Cas98minus6

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Cas98minus7

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Cas98minus8

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leminus20

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Cas98minus9

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leminus20

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minus10 minus5 0 5 10 15

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Cas98minus10

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leminus20

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-30

-118-33

Cou

nts

Indel size [bp]

Supplementary Figure 5 Size distribution of indels at the on-target site of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions When deletions are smaller than 20 bp the actual deletion sizes are indicated Note that Cas98-4 has a 118-bp deletion Its read count is much lower than the rest because the corresponding small PCR products were depleted during size selection

Nature Biotechnology doi101038nbt2800

Supplementary Figure 6 A competition assay between wild type and Piga-deficient ESCs ab Flow cytom-etry analyses Piga-deficient ESCs were obtained by alpha-toxin selection of hCas9-expressing ESCs trans-fected with the Piga Site 2 gRNA expression vector Wild type and mutant ESCs were mix at a 11 ratio and cultured under feeder-free conditions in 4 replicates Flow cytometry analyses using FLAER were performed at every passage The Piga-deficient (FLAER-negative) ESC population was depleted over time Data are shown as mean plusmn sd (n=4)

a P1 P2 P3 P4 P5 P6

b

0

10

20

30

40

50

60

70

P1 P2 P3 P4 P5 P6

FLA

ER

-neg

ativ

e (

)

Passage

Nature Biotechnology doi101038nbt2800

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0

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leminus20

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Cas95minus1

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Cas95minus2

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Cas95minus3

0

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leminus20

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Cas95minus4

0

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2000

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leminus20

minus15

minus10 minus5 0 5 10 15

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Cas95minus5

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

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Cas95minus6

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus7

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

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Cas95minus8

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus9

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus10

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus1

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus2

0

1000

2000

3000

4000

5000

leminus20

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minus10 minus5 0 5 10 15

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Cas98minus3

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

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Cas98minus4

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

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Cas98minus5

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus6

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus7

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus8

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

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Cas98minus9

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

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Cas98minus10

0

1000

2000

3000

4000

5000

leminus20

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minus10 minus5 0 5 10 15

ge20

Cou

nts

Indel size [bp]

Supplementary Figure 7 Size distribution of indels at the off-target site (l20_5tm-21) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

JM8

0

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leminus20

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Cas95minus1

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leminus20

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Cas95minus2

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Cas95minus3

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leminus20

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Cas95minus4

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Cas95minus5

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Cas95minus6

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Cas95minus7

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Cas98minus9

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Cas98minus10

0

500

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leminus20

minus15

minus10 minus5 0 5 10 15

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Cou

nts

Indel size [bp]

Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

0

20

40

60

80

100

2 8Days post infection

p=00003

Piga Site 3 endogenousPiga Site 1 endogenousPiga Site 2 endogenousPiga Site 3 alteredPiga Site 1 alteredEmpty vectorMock infection

BFP

-pos

itive

()

Nature Biotechnology doi101038nbt2800

Dpm1 (1)

Pgap2 (2)

Pigg (1)

Pign (1)

Pigt (1)

Pigy (1)

Dpm2 (1)

Piga (2)

Pigh (1)

Pigo (1)

Pigu (1)

Control

Dpm3 (1)

Pigb (1)

Pigk (1)

Pigp (1)

Pigv (2)

Gpaa1 (1)

Pigc (2)

Pigl (2)

Pigq (1)

Pigw (2)

Mpdu1 (1)

Pigf (2)

Pigm (2)

Pigs (1)

Pigx (2)

Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

BFP

Nature Biotechnology doi101038nbt2800

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∆7bp

∆7bp

∆5bp

∆11bp∆4bp

∆2bp∆2bp ∆2bp

∆12bp

∆9bp

∆6bp

∆6bp∆9bp

∆8bp

∆5bp

∆11bp

∆18bp

∆11bp

∆6bp ∆6bp

∆6bp

∆6bp∆6bp

∆6bp

∆6bp∆15bp

∆15bp

∆5bp∆10bp

Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Rea

d co

unts

Indel size [bp]

Nature Biotechnology doi101038nbt2800

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Dpm1

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Pigu

minus40 minus20 0 20 40

0

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minus40 minus20 0 20 40

0

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Pigw

minus40 minus20 0 20 40

0

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3000

4000

5000

Pigx

minus40 minus20 0 20 40

Pigy

∆7bp

∆7bp∆7bp

∆7bp

∆7bp∆17bp

∆7bp

∆4bp ∆4bp ∆2bp

∆5bp

∆5bp

∆5bp ∆6bp ∆6bp

∆5bp

∆6bp ∆6bp ∆6bp

∆14bp

∆12bp

∆13bp ∆10bp ∆10bp∆14bp

NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

Pgap2

Pigg Pign

Pigt

Pigy

Dpm2

Piga

Dpm3

Pigb

Gpaa1

Pigc

Mpdu1

Pigf

Pigk Pigl Pigm

Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

0

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Pgap2

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Pigm

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Pigp

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Pigx

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2000

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1000

0

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1000

Indel size [bp]

Rea

d co

unts

Nature Biotechnology doi101038nbt2800

0

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40

60

80

100

120

Dpm

1D

pm2

Dpm

3G

paa1

Mpd

u1P

gap2

Pig

aP

igb

Pig

cP

igf

Pig

gP

igh

Pig

kP

igl

Pig

mP

ign

Pig

oP

igp

Pig

qP

igs

Pig

tP

igu

Pig

vP

igw

Pig

xP

igy

Perc

enta

ge

Ins+Del

Ins

Del

0

20

40

60

80

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120

Dpm

1D

pm2

Dpm

3G

paa1

Mpd

u1P

gap2

Pig

aP

igb

Pig

cP

igf

Pig

gP

igh

Pig

kP

igl

Pig

mP

ign

Pig

oP

igp

Pig

qP

igs

Pig

tP

igu

Pig

vP

igw

Pig

xP

igy

Perc

enta

ge

Ins+Del

Ins

Del

a

b

NA

NA

Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

NA

gRNA

Dep

letio

n ra

te re

lativ

eto

the

lent

ivira

l lib

rary

Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

gRN

A si

te 1

gRN

A si

te 1

Mlh1Msh6

TRC

N00

0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

Moc

k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 5: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Site4Site2

Site3 Site1

Site 1 TCACTCCAGACCCATTTAGG AGGSite 2 GAAGAGAGCATCATGGGCCA TGGSite 3 CGGATTTGCTGATGTCAGCT CGGSite 4 CAGAGAAAGAACTGTGGGAA TGG

Piga

U6 gRNA scaffold Terminatorspacer

GGACGAAACACCGGGTCTTCGAGAAGACGTTTTAGAGCTAGACCTGCTTTGTGGCCCAGAAGCTCTTCTGCAAAATCTCGATCT

Transciption

5rsquo-CACC NNNNN NNNNN NNNNN NNNNN GT-3rsquo 3rsquo-nnnnn nnnnn nnnnn nnnnn CAAAAT-5rsquo

Duplex oligos

a

d

b

FLA

ER

-neg

ativ

e ce

lls (

)

FSC

FLA

ER

cSite 2

Site 3 No gRNA

Site 1

Supplementary Figure 3 Construction and validation of gRNA-expression vector a Schematic of the gRNA cloning vector BbsI digestion removes the spacer and produces cohesive ends which allows duplex oligonu-cleotides with compatible overhangs to ligate into Underlined BbsI sites For the duplex oligonucleotides compatible overhangs are shown in blue gRNA sequences are shown as N (top strand) and n (bottom strand) b The sequences and the genomic positions of gRNAs targeting the Piga gene The genomic PAM sequence of each gRNA is shown in red c Flow cytometry analysis of GPI-anchored protein expression Analysis was performed 6 days post transfection d A summary of the flow cytometry analyses in c Transfec-tion was performed in triplicates Data are shown as mean plusmn sd (n=3)

Site 4

0

5

10

15

20

Site 1 Site 2 Site 3 Site 4 No gRNA

Nature Biotechnology doi101038nbt2800

a bU6 20-bp

guide seqgRNA scaffold T7PB PBPGKneopA

MscI

12 kb

30

506080100

20

15

10

05

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

JM8-Cas5 JM8-Cas8

Supplementary Figure 4 Generation of doubly transgenic ESC lines a Schematic of the piggyBac transpo-son vector carrying the gRNA expression cassette together with the G418 resistant gene cassette The thick line represents the probe for Southern blot analysis b Southern blot analysis of gRNA-expressing ESC clones Genomic DNA was digested with MscI and hybridized with the radiolabeled probe shown in a Note that the shortest band size is 12kb All clones analysed carry a single gRNA expression cassette

kb

Nature Biotechnology doi101038nbt2800

JM8

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus1

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus2

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus3

0

500

1000

1500

2000

2500

3000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus4

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus5

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus6

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus7

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus8

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus9

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus10

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus1

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus2

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus3

0

500

1000

1500

2000

2500

3000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus4

0

100

200

300

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus5

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus6

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus7

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus8

0

5000

10000

15000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus9

0

500

1000

1500

2000

2500

3000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus10

0

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Supplementary Figure 5 Size distribution of indels at the on-target site of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions When deletions are smaller than 20 bp the actual deletion sizes are indicated Note that Cas98-4 has a 118-bp deletion Its read count is much lower than the rest because the corresponding small PCR products were depleted during size selection

Nature Biotechnology doi101038nbt2800

Supplementary Figure 6 A competition assay between wild type and Piga-deficient ESCs ab Flow cytom-etry analyses Piga-deficient ESCs were obtained by alpha-toxin selection of hCas9-expressing ESCs trans-fected with the Piga Site 2 gRNA expression vector Wild type and mutant ESCs were mix at a 11 ratio and cultured under feeder-free conditions in 4 replicates Flow cytometry analyses using FLAER were performed at every passage The Piga-deficient (FLAER-negative) ESC population was depleted over time Data are shown as mean plusmn sd (n=4)

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 7 Size distribution of indels at the off-target site (l20_5tm-21) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

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Nature Biotechnology doi101038nbt2800

Dpm1 (1)

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Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

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Nature Biotechnology doi101038nbt2800

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NA

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Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

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Pighwt Pigh∆12 Control

FLAER

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Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

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Ncoa3Nanog

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Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

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HscbH2minusKe2Gtpbp4

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Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

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metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

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Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

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Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

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Mlh1 Wt

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Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 6: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

a bU6 20-bp

guide seqgRNA scaffold T7PB PBPGKneopA

MscI

12 kb

30

506080100

20

15

10

05

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

JM8-Cas5 JM8-Cas8

Supplementary Figure 4 Generation of doubly transgenic ESC lines a Schematic of the piggyBac transpo-son vector carrying the gRNA expression cassette together with the G418 resistant gene cassette The thick line represents the probe for Southern blot analysis b Southern blot analysis of gRNA-expressing ESC clones Genomic DNA was digested with MscI and hybridized with the radiolabeled probe shown in a Note that the shortest band size is 12kb All clones analysed carry a single gRNA expression cassette

kb

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 5 Size distribution of indels at the on-target site of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions When deletions are smaller than 20 bp the actual deletion sizes are indicated Note that Cas98-4 has a 118-bp deletion Its read count is much lower than the rest because the corresponding small PCR products were depleted during size selection

Nature Biotechnology doi101038nbt2800

Supplementary Figure 6 A competition assay between wild type and Piga-deficient ESCs ab Flow cytom-etry analyses Piga-deficient ESCs were obtained by alpha-toxin selection of hCas9-expressing ESCs trans-fected with the Piga Site 2 gRNA expression vector Wild type and mutant ESCs were mix at a 11 ratio and cultured under feeder-free conditions in 4 replicates Flow cytometry analyses using FLAER were performed at every passage The Piga-deficient (FLAER-negative) ESC population was depleted over time Data are shown as mean plusmn sd (n=4)

a P1 P2 P3 P4 P5 P6

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 7 Size distribution of indels at the off-target site (l20_5tm-21) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

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leminus20

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Cas95minus3

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minus10 minus5 0 5 10 15

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minus10 minus5 0 5 10 15

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Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

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Piga Site 3 endogenousPiga Site 1 endogenousPiga Site 2 endogenousPiga Site 3 alteredPiga Site 1 alteredEmpty vectorMock infection

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Nature Biotechnology doi101038nbt2800

Dpm1 (1)

Pgap2 (2)

Pigg (1)

Pign (1)

Pigt (1)

Pigy (1)

Dpm2 (1)

Piga (2)

Pigh (1)

Pigo (1)

Pigu (1)

Control

Dpm3 (1)

Pigb (1)

Pigk (1)

Pigp (1)

Pigv (2)

Gpaa1 (1)

Pigc (2)

Pigl (2)

Pigq (1)

Pigw (2)

Mpdu1 (1)

Pigf (2)

Pigm (2)

Pigs (1)

Pigx (2)

Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

BFP

Nature Biotechnology doi101038nbt2800

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∆7bp

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∆12bp

∆9bp

∆6bp

∆6bp∆9bp

∆8bp

∆5bp

∆11bp

∆18bp

∆11bp

∆6bp ∆6bp

∆6bp

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∆6bp

∆6bp∆15bp

∆15bp

∆5bp∆10bp

Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Rea

d co

unts

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Nature Biotechnology doi101038nbt2800

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∆7bp∆7bp

∆7bp

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∆7bp

∆4bp ∆4bp ∆2bp

∆5bp

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∆5bp ∆6bp ∆6bp

∆5bp

∆6bp ∆6bp ∆6bp

∆14bp

∆12bp

∆13bp ∆10bp ∆10bp∆14bp

NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

Pgap2

Pigg Pign

Pigt

Pigy

Dpm2

Piga

Dpm3

Pigb

Gpaa1

Pigc

Mpdu1

Pigf

Pigk Pigl Pigm

Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

NA

gRNA

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Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

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2844

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4996

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2494

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Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 7: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

JM8

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Cas95minus3

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Cas95minus4

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Cas95minus5

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Cas95minus6

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Cas95minus7

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Cas95minus8

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minus10 minus5 0 5 10 15

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Cas95minus9

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Cas98minus6

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Cas98minus7

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Cas98minus8

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Cas98minus9

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Cas98minus10

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15000

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minus10 minus5 0 5 10 15

ge20

-30

-118-33

Cou

nts

Indel size [bp]

Supplementary Figure 5 Size distribution of indels at the on-target site of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions When deletions are smaller than 20 bp the actual deletion sizes are indicated Note that Cas98-4 has a 118-bp deletion Its read count is much lower than the rest because the corresponding small PCR products were depleted during size selection

Nature Biotechnology doi101038nbt2800

Supplementary Figure 6 A competition assay between wild type and Piga-deficient ESCs ab Flow cytom-etry analyses Piga-deficient ESCs were obtained by alpha-toxin selection of hCas9-expressing ESCs trans-fected with the Piga Site 2 gRNA expression vector Wild type and mutant ESCs were mix at a 11 ratio and cultured under feeder-free conditions in 4 replicates Flow cytometry analyses using FLAER were performed at every passage The Piga-deficient (FLAER-negative) ESC population was depleted over time Data are shown as mean plusmn sd (n=4)

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Supplementary Figure 7 Size distribution of indels at the off-target site (l20_5tm-21) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

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Nature Biotechnology doi101038nbt2800

Dpm1 (1)

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Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

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Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

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Srfbp1Snrpb

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Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

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Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

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E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

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Tcp1Rrn3Rps5Rps3

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Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

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metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

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VhlPsmc4Psmc3Mdm2

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1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

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Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

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Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 8: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Supplementary Figure 6 A competition assay between wild type and Piga-deficient ESCs ab Flow cytom-etry analyses Piga-deficient ESCs were obtained by alpha-toxin selection of hCas9-expressing ESCs trans-fected with the Piga Site 2 gRNA expression vector Wild type and mutant ESCs were mix at a 11 ratio and cultured under feeder-free conditions in 4 replicates Flow cytometry analyses using FLAER were performed at every passage The Piga-deficient (FLAER-negative) ESC population was depleted over time Data are shown as mean plusmn sd (n=4)

a P1 P2 P3 P4 P5 P6

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 7 Size distribution of indels at the off-target site (l20_5tm-21) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

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Nature Biotechnology doi101038nbt2800

Dpm1 (1)

Pgap2 (2)

Pigg (1)

Pign (1)

Pigt (1)

Pigy (1)

Dpm2 (1)

Piga (2)

Pigh (1)

Pigo (1)

Pigu (1)

Control

Dpm3 (1)

Pigb (1)

Pigk (1)

Pigp (1)

Pigv (2)

Gpaa1 (1)

Pigc (2)

Pigl (2)

Pigq (1)

Pigw (2)

Mpdu1 (1)

Pigf (2)

Pigm (2)

Pigs (1)

Pigx (2)

Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

BFP

Nature Biotechnology doi101038nbt2800

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∆7bp

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∆9bp

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Rea

d co

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Indel size [bp]

Nature Biotechnology doi101038nbt2800

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NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

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Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Pigx

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0

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2000

0

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0

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1000

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Rea

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Nature Biotechnology doi101038nbt2800

0

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120

Dpm

1D

pm2

Dpm

3G

paa1

Mpd

u1P

gap2

Pig

aP

igb

Pig

cP

igf

Pig

gP

igh

Pig

kP

igl

Pig

mP

ign

Pig

oP

igp

Pig

qP

igs

Pig

tP

igu

Pig

vP

igw

Pig

xP

igy

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enta

ge

Ins+Del

Ins

Del

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Dpm

1D

pm2

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paa1

Mpd

u1P

gap2

Pig

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cP

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gP

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NA

NA

Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

NA

gRNA

Dep

letio

n ra

te re

lativ

eto

the

lent

ivira

l lib

rary

Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

gRN

A si

te 1

gRN

A si

te 1

Mlh1Msh6

TRC

N00

0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

Moc

k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 9: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

JM8

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus1

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus2

0

1000

2000

3000

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5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus3

0

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2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus4

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus5

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus6

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus7

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus8

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus9

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus10

0

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leminus20

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ge20

Cas98minus1

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leminus20

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minus10 minus5 0 5 10 15

ge20

Cas98minus2

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leminus20

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minus10 minus5 0 5 10 15

ge20

Cas98minus3

0

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leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus4

0

1000

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leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus5

0

1000

2000

3000

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5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus6

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus7

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus8

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus9

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus10

0

1000

2000

3000

4000

5000

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cou

nts

Indel size [bp]

Supplementary Figure 7 Size distribution of indels at the off-target site (l20_5tm-21) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

JM8

0

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1500

leminus20

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Cas95minus1

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leminus20

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Cas95minus2

0

500

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1500

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus3

0

500

1000

1500

leminus20

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minus10 minus5 0 5 10 15

ge20

Cas95minus4

0

500

1000

1500

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus5

0

500

1000

1500

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus6

0

500

1000

4400

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus7

0

500

1000

1500

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus8

0

500

1000

1500

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus9

0

500

1000

1500

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas95minus10

0

500

1000

1500

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus1

0

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2800

leminus20

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minus10 minus5 0 5 10 15

ge20

Cas98minus2

0

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leminus20

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minus10 minus5 0 5 10 15

ge20

Cas98minus3

0

500

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1500

leminus20

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minus10 minus5 0 5 10 15

ge20

Cas98minus4

0

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1000

1500

leminus20

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minus10 minus5 0 5 10 15

ge20

Cas98minus5

0

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1000

1500

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus6

0

500

1000

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leminus20

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minus10 minus5 0 5 10 15

ge20

Cas98minus7

0

500

1000

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leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cas98minus8

0

500

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7000

leminus20

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minus10 minus5 0 5 10 15

ge20

Cas98minus9

0

500

1000

1500

leminus20

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minus10 minus5 0 5 10 15

ge20

Cas98minus10

0

500

1000

6100

leminus20

minus15

minus10 minus5 0 5 10 15

ge20

Cou

nts

Indel size [bp]

Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

0

20

40

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p=00003

Piga Site 3 endogenousPiga Site 1 endogenousPiga Site 2 endogenousPiga Site 3 alteredPiga Site 1 alteredEmpty vectorMock infection

BFP

-pos

itive

()

Nature Biotechnology doi101038nbt2800

Dpm1 (1)

Pgap2 (2)

Pigg (1)

Pign (1)

Pigt (1)

Pigy (1)

Dpm2 (1)

Piga (2)

Pigh (1)

Pigo (1)

Pigu (1)

Control

Dpm3 (1)

Pigb (1)

Pigk (1)

Pigp (1)

Pigv (2)

Gpaa1 (1)

Pigc (2)

Pigl (2)

Pigq (1)

Pigw (2)

Mpdu1 (1)

Pigf (2)

Pigm (2)

Pigs (1)

Pigx (2)

Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

BFP

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

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Nature Biotechnology doi101038nbt2800

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NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

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Pighwt Pigh∆12 Control

FLAER

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Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

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Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

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Ext2 (Site 2)

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Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

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Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 10: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

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Supplementary Figure 8 Size distribution of indels at the off-target site (l20_5tm-2) of the gRNA targeting Piga Site 2 Plus values and blue bars represent insertions while minus values and red bars represent deletions

Nature Biotechnology doi101038nbt2800

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

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Nature Biotechnology doi101038nbt2800

Dpm1 (1)

Pgap2 (2)

Pigg (1)

Pign (1)

Pigt (1)

Pigy (1)

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Control

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Pigc (2)

Pigl (2)

Pigq (1)

Pigw (2)

Mpdu1 (1)

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Pigm (2)

Pigs (1)

Pigx (2)

Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

BFP

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

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Nature Biotechnology doi101038nbt2800

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NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

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Pigc

Mpdu1

Pigf

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Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

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Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

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1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

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Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 11: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Supplementary Figure 9 Proviral silencing BFP-positve fractions were measured at 2 and 8 days post infection Data shown is from one of the duplicated experiments Similar silencing was observed in the duplicated experiment Paired t-test was performed

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Nature Biotechnology doi101038nbt2800

Dpm1 (1)

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Pigl (2)

Pigq (1)

Pigw (2)

Mpdu1 (1)

Pigf (2)

Pigm (2)

Pigs (1)

Pigx (2)

Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

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Nature Biotechnology doi101038nbt2800

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∆7bp

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Rea

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Nature Biotechnology doi101038nbt2800

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∆5bp

∆6bp ∆6bp ∆6bp

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∆13bp ∆10bp ∆10bp∆14bp

NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

Pgap2

Pigg Pign

Pigt

Pigy

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Piga

Dpm3

Pigb

Gpaa1

Pigc

Mpdu1

Pigf

Pigk Pigl Pigm

Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

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Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

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Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

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Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 12: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Dpm1 (1)

Pgap2 (2)

Pigg (1)

Pign (1)

Pigt (1)

Pigy (1)

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Pigo (1)

Pigu (1)

Control

Dpm3 (1)

Pigb (1)

Pigk (1)

Pigp (1)

Pigv (2)

Gpaa1 (1)

Pigc (2)

Pigl (2)

Pigq (1)

Pigw (2)

Mpdu1 (1)

Pigf (2)

Pigm (2)

Pigs (1)

Pigx (2)

Supplementary Figure 10 Flow cytometry analysis of ESCs transfected with the indicated gRNA expression vectors These are the flow cytometry profiles that are summarised in Figure 3b The profiles from the gRNAs with higher FLAER-negative values are shown for each of the 26 GPI pathway genes Note that Pigf Pign Pigq and Pigw knockouts do not completely lose the expression of GPI-anchored proteins instead express-ing them at a reduced level

FLAER

BFP

Nature Biotechnology doi101038nbt2800

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

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Nature Biotechnology doi101038nbt2800

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∆5bp

∆6bp ∆6bp ∆6bp

∆14bp

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∆13bp ∆10bp ∆10bp∆14bp

NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

Pgap2

Pigg Pign

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Pigy

Dpm2

Piga

Dpm3

Pigb

Gpaa1

Pigc

Mpdu1

Pigf

Pigk Pigl Pigm

Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

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Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

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2844

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4996

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Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 13: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

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∆6bp ∆6bp

∆6bp

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∆6bp

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Supplementary Figure 11 Size distribution of indels at Site 1 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

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Nature Biotechnology doi101038nbt2800

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∆5bp

∆5bp

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∆5bp

∆6bp ∆6bp ∆6bp

∆14bp

∆12bp

∆13bp ∆10bp ∆10bp∆14bp

NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

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Pigk Pigl Pigm

Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

NA

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Dep

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Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

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7713

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Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 14: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

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∆5bp

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NA

NA

Supplementary Figure 12 Size distribution of indels at Site 2 of each gene Deletions with clear peaks are often associated with micro-homology These deletions are marked with their deletion sizes The sequences of these sites are summarised in Supplementary Table 4

Nature Biotechnology doi101038nbt2800

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

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Pighwt Pigh∆12 Control

FLAER

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Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

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Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

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Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

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ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

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Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 15: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Supplementary Figure 13 Functional analysis of the Pigh∆12 gene product hCas9-expressing ESCs were transfected with the Pigh Site 1 gRNA expression vector and FLAER-negative cells were isolated by cell sorting Subsequently these cells were individually transfected with cDNA expressing each of the 26 GPI-anchor-biosynthesis pathway genes or Pigh∆12 Two days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Note that the Pigh∆12 gene product can restore the expression of GPI-anchored proteins as efficiently as wild type Pigh

Dpm1

Pgap2

Pigg Pign

Pigt

Pigy

Dpm2

Piga

Dpm3

Pigb

Gpaa1

Pigc

Mpdu1

Pigf

Pigk Pigl Pigm

Pigo Pigp Pigq Pigs

Pigu Pigv Pigw Pigx

Pighwt Pigh∆12 Control

FLAER

BFP

Nature Biotechnology doi101038nbt2800

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Pigx

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Nature Biotechnology doi101038nbt2800

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Dpm

1D

pm2

Dpm

3G

paa1

Mpd

u1P

gap2

Pig

aP

igb

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cP

igf

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gP

igh

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kP

igl

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mP

ign

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igp

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igs

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enta

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Ins+Del

Ins

Del

a

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NA

NA

Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

NA

gRNA

Dep

letio

n ra

te re

lativ

eto

the

lent

ivira

l lib

rary

Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

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te 1

gRN

A si

te 1

Mlh1Msh6

TRC

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0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

Moc

k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 16: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Supplementary Figure 14 Reproducibility of micro-homology mediated repair Size distributions of indels at Site 1 of the indicated genes from 4 independent experiments are shown Reproducibility was observed at all sites

Experiment 1 Experiment 2 Experiment 3 Experiment 4

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Nature Biotechnology doi101038nbt2800

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

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Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

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Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

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Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 17: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

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Supplementary Figure 15 Mutation signatures associated with CRISPR-Cas9-induced DSBs ab Signatures at Site 1(a) and Site 2 (b) of the indicated genes Mutated sequences are clasified into three groups insertion only deletion only and insertion+deletion Data are shown as mean plusmn sd (n=2-4)

Nature Biotechnology doi101038nbt2800

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

NA

gRNA

Dep

letio

n ra

te re

lativ

eto

the

lent

ivira

l lib

rary

Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

gRN

A si

te 1

gRN

A si

te 1

Mlh1Msh6

TRC

N00

0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

Moc

k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 18: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Zfp574Wdr55Wdr36Vps72Usp39

TwistnbTsr2

Tcf15Tbl3

TardbpSrsf2Srsf1

Srfbp1Snrpb

Smad7Sf3b5Sf3b3

Sf1SbdsRrp9

Rpl26Rpf1

Rbbp4Ppp1r10

Pop5Polr2jPolr1cPolr1aPmf1

Phf5aPes1Pelp1

Nrf1Nop2

Ncoa3Nanog

Imp4Imp3

Hspa8Hinfp

Gtf3c4Gemin5

Ftsj3Exosc5Exosc3Eif4a3Eftud2

EcdE4f1Dkc1

Ddx56Ddx54

Clp1Cirh1aAlyrefRNA processing

ncRNA metabolic process p=46E-20RNA processing p=28E-19rRNA processing p=18E-18rRNA metabolic process p=21E-18ncRNA processing p=82E-17RNA splicing p=81E-05mRNA processing p=10E-04tRNA metabolic process p=15E-04mRNA metabolic process p=60E-04 RNA biosynthetic process p=20E-03transcription DNA-dependent p=27E-02

Rrm2Rrm1Rpa1Rfc5

Rbbp4Pold1PcnaOrc6

Mcm7Mcm6Mcm5Mcm3Mcm2Gins4Gins2

E4f1Dna2Cinp

Brca1DNA replicationDNA replication p=69E-13DNA-dependent DNA replication p=22E-07DNA replication initiation p=67E-04DNA unwinding during replication p=22E-02DNA duplex unwinding p=28E-02DNA geometric change p=31E-02DNA metabolic process p=31E-08

Usp39Rbbp4

Ppp1cbPdcd2lMcm7Mcm6Mcm3Mcm2Hcfc1

Gnb2l1Cinp

Chmp3Arl2

Rpl13aRpl10Pfdn2Pes1

Mrpl20Hspa9Hspa8

HscbH2minusKe2Gtpbp4

Gnl2Cct7Cct5Cct4Cct3Cct2

Tcp1Rrn3Rps5Rps3

Rps20Rplp0

Rpl8Rpl6Rpl4

Rpl36aRpl26Rpl19Rpl18

Protein synthesisribosome biogenesis p=12E-22ribonucleoprotein complex biogenesis p=14E-21translation p=10E-12translation initiation p=15E-03tRNA aminoacylation for protein translation p=28E-02amino acid activation p=28E-02tRNA aminoacylation p=28E-02protein folding p=20E-03

Nubp1NarflIscu

HscbFxnCellular respiration

metallo-sulfur cluster assembly p=67E-04iron-sulfur cluster assembly p=67E-04

Cell cyclecell cycle p=73E-05mitotic cell cycle p=85E-03cell cycle phase p=31E-02

VhlPsmc4Psmc3Mdm2

Psmc4Psmc3Pou5f1

Protein degradationprotein catabolic process p=14E-02macromolecule catabolic process p=19E-02

Blastocyst developmentblastocyst development p=49E-02

1gRNA 2 3 4 5 1 2 3 4 5ESC lib1 ESC lib2

1 2 3 4 5 1 2 3 4 5

ESC lib1 ESC lib2

Supplementary Figure 16 Gene ontology analyses of the depleted genes Genes that are depleted in the ESC libraries were used for gene ontology analysis GO terms that are overrepresented with the depleted genes and genes associated with these GO terms are shown Heatmaps show the depletion rate of each gRNA in ESC libraries 1 and 2 NA not applicable since gRNAs were not designed

1000100101010010001

NA

gRNA

Dep

letio

n ra

te re

lativ

eto

the

lent

ivira

l lib

rary

Nature Biotechnology doi101038nbt2800

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

gRN

A si

te 1

gRN

A si

te 1

Mlh1Msh6

TRC

N00

0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

Moc

k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 19: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Piga (site 3)

No gRNA

B4galt7 (Site 4)

Ext2 (Site 3)

B4galt7 (Site 3)

Ext2 (Site 2)

1700016K19Rik (Site 4)1700016K19Rik (Site 3)

Cstf3 (Site 2)Cstf3 (Site 1)

FSC

FLA

ER

Supplementary Figure 17 Flow cytometry analysis of GPI-anchored protein expression in ESCs transfected with the gRNA expression vectors targeting the candidate genes hCas9-expressing ESCs were individually transfected with the vectors expressing the indicated gRNAs Six days post transfection the cells were stained with FLAER and anlaysed by flow cytometry Inactivation of these 4 candidate genes increased resistance to alpha-toxin (Fig 5c) Inactivation of the genes however did not affect GPI-anchored protein expression

Nature Biotechnology doi101038nbt2800

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

gRN

A si

te 1

gRN

A si

te 1

Mlh1Msh6

TRC

N00

0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

Moc

k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 20: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Letmd1

GM15293

GM15293 Prkg1

Mlh1 Wt

6TG-treated(2 microM)

Non-treated

Supplementary Figure 18 Validation of the candidate genes Methylene blue staining of ESCs Cas9-expressing ESCs were transfected with vectors expressing gRNAs targeting the indicated genes These cells were subsequently treated with 6TG The treatment was performed in 12-well plates Different gRNAs were used in each well and these are marked accordingly on the right panel The gRNAs targeting Mlh1 were used as a positive control in each treatment

Nature Biotechnology doi101038nbt2800

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

gRN

A si

te 1

gRN

A si

te 1

Mlh1Msh6

TRC

N00

0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

Moc

k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 21: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

TRC

N00

0030

2844

TRC

N00

0030

4996

TRC

N00

0030

5029

TRC

N00

0033

7644

TRC

N00

0033

7646

TRC

N00

0033

7713

TRC

N00

0033

7714

gRN

A si

te 1

gRN

A si

te 1

gRN

A si

te 2

Piga Pigx

alpha-toxin-treated

Non-treated

Em

pty

viru

s

Moc

k-in

fect

ed

a

b

gRN

A si

te 1

gRN

A si

te 1

Mlh1Msh6

TRC

N00

0030

3112

TRC

N00

0004

2721

gRN

A si

te 1

Pms2

TRC

N00

0024

0634

TRC

N00

0024

0635

Msh2

gRN

A si

te 1

TRC

N00

0004

2494

Em

pty

viru

s

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k-in

fect

ed

6TG-treated

Non-treated

Supplementary Figure 19 Comparison between gRNA-mediated knockout and shRNA-mediated knock-down a-b Methylene blue staining of ESCs Cas9-expressing ESCs were infected with lentiviruses expressing the indicated gRNA or shRNA and treated with 10 nM alpha-toxin (a) or 2 microM 6TG (b) Alpha-toxin treatment was performed in 96-well plates 6TG treatment was performed in 12-well plates Except one shRNA target-ing Mlh1 all shRNAs were not able to produce resistant cells at a level comparable to the corresponding gRNAs

Nature Biotechnology doi101038nbt2800

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

Page 22: Supplementary Results Deep sequencing analyses of on ... · Supplementary Results . Deep sequencing analyses of on-target cleavages at a clonal level. ... Pigp Pigq PIgy Dpm2 Dpm1

Supplementary Table 3 List of the most frequent deletion with a micro-homology

Gene Site Guide sequence PAM 3 flanking Deletion size [bp]

Dpm1 Site 1 GATGATCACATAGTTCC|CCG TGG CGTGTTT Δ7

Dpm2 Site 1 GGAGGAGGACGGCGTAG|GCG CGG GGCAGGA Δ6

Dpm3 Site 1 GCCTACCTGTTGGTCTC|CGC TGG CTGCTAT Δ10

Gpaa1 Site 1 GTCCCTCCACTGTCACA|TCG AGG CTGGTGA Δ5

Mpdu1 Site 1 GATGCTGTAGACCACGG|TTC CGG TCAGTGC Δ6

Pgap2 Site 1 GCCGGACTAGGGTCCCA|TCC CGG TCCAATG Δ11

Piga Site 1 TCACTCCAGACCCATTT|AGG AGG CATGATA Δ11

Pigb Site 1 GTAAATACTGGCGAAGA|TGA GGG GGTAAGT No strong signal

Pigc Site 1 GTTTTATTCGATCTTAT|CGA TGG AGGTGAT Δ9

Pigf Site 1 CTCAGCCATGCTTTGAG|GTT TGG TCCTAAC Δ7

Pigg Site 1 GATTTTGTGTTTGGGTC|GAA AGG TGTGAAG Δ8

Pigh Site 1 GGCCGCCAGCCACACCG|CGC AGG TGACCGC Δ2

Pigk Site 1 GTATGGAGACGATGTGG|AAG TGG ACTACAG Δ6

Pigl Site 1 TCTGCAAAGCTGTGCTG|TGT TGG GGATTCC Δ5

Pigm Site 1 GTCTACCTCAGCGAACT|CTT CGG CAAGTTT Δ2

Pign Site 1 GAGCAGGTAGGTGGACA|TCA AGG AGAGGAG Δ9

Pigo Site 1 GTGCTGATCGCTCACTT|CCT GGG TGTGGAT Δ4

Pigp Site 1 GAATTCTCTTGTGCAGA|TAC TGG GCGGTTG Δ11

Pigq Site 1 GTCAGCTAGGCCTCCGG|TGC TGG CTGTGGT Δ6

Pigs Site 1 GATCTCTCGCTCATGCA|CAA CGG TGAAAGG Δ2

Pigt Site 1 GCTTTTGGAGGACGCGA|TAC TGG GGGCCAC Δ15

Pigu Site 1 GAGGGTGTTGTTGATGG|CGC AGG TAGACTT Δ6

Pigv Site 1 GCTGATCCGCAGAGCCC|GAG GGG GCGAGGC Δ6

Pigw Site 1 GGCTATTGCCATTTTGG|CCG TGG ATTTCCC Δ6

Pigx Site 1 CGTAGTGAAGCCAACTC|ATA GGG ATCCACA Δ15

Pigy Site 1 GCGCACTGTCTGCAGTC|GCC GGG AGGTTTC Δ6

Dpm1 Site 2 GCTTTTTCTCTCGTGGT|CTT AGG AGCTTCA Δ7

Dpm2 Site 2 ACAGACCAAGCAGTAGG|ATT TGG CCTTGTC Δ6

Dpm3 Site 2 GACCATGGGAGCACTGG|GCT TGG AGTTGCC Δ6

Gpaa1 Site 2 GCCCCAGGCTCCCAAGC|GGG TGG TCCCACC Δ14

Mpdu1 Site 2 GGAAGCAGATGGCAACG|GTC TGG AGTGTCA NA

Pgap2 Site 2 GAAACGCCACACGTAGC|GTT GGG GGACCTC Δ5

Piga Site 2 GAAGAGAGCATCATGGG|CCA TGG CAGAGAA Δ7

Pigb Site 2 GTTAAGTATTCGCAGAG|CGA TGG TAAACAA Δ4

Pigc Site 2 GTCACTCCACGCCTTCA|TTA TGG TGACGTT Δ4

Pigf Site 2 ATAGAACAAAAATGATG|TGA AGG AGAAAAC Δ2

Pigg Site 2 GGGCCCAACAGCCCCCT|GAT TGG TCACAAG No strong signal

Pigh Site 2 GCCTAAGATGGTGATGA|AGA TGG TGGCAGA Δ12

Pigk Site 2 GAAAGAAGACGCTTTGA|GCG AGG GGTACTG Δ7

Pigl Site 2 GTGTAATGATTATTGAC|AAG AGG TAATTTG Δ5

Pigm Site 2 GGTAGGTCGCCCGCAGG|TAC GGG GAGCGCC Δ5

Pign Site 2 GAGGAAAGGGGACTCCA|ATA AGG GATGCCA Δ13

Pigo Site 2 GACCACCATGCAGCGTC|TAA AGG CTCTCAC Δ10

Pigp Site 2 GGGCTTTCGTTCCCGAA|TCT TGG TTAAACT Δ7

Pigq Site 2 GACTGGAGGCGGATGCA|ACC AGG AGAGCAG Δ17

Pigs Site 2 GAAGGCCTACCGGAGAG|CTT TGG AGCACGA Δ10

Pigt Site 2 GAGGCAGCACAGCGTAG|CGC AGG AAGTAGT Δ5

PigU Site 2 AGGGATGTAGCGCATTT|CCA TGG GGGTTCG Δ6

Pigv Site 2 GCAGTGCTGCAGCGTGG|ACC AGG TACACAA Δ6

Pigw Site 2 GCTGTTAATGACCCGGT|AAC AGG TGATTGC Δ7

Pigx Site 2 GGATCCACAAAGAGTCC|TGG TGG AATGTAA No strong signal

Pigy Site 2 ACCAGGAACTGCAGCAG|CGC CGG GTGGAAG NA

Predicted cut sites are shown as |

Deleted sequences are shown in bold letters

Micro-homologies at either side of the breaks are underlined

Sequence

Nature Biotechnology doi101038nbt2800

Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

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Supplementary Table 4 Accuracy of guide RNA sequences in the whole-genome lentiviral library

Clones with correct sequences

Clones with incorrect sequences

Point mutation(s) 5 (36 )

Indel(s) 6 (43 )

Mutations in the 3 flanking region 7 (50 )

Total clones analyzed

121 (871 )

18 (129 )

139

Nature Biotechnology doi101038nbt2800

Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

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Supplementary Table 5 GO analyses of the genes depleted in the genome-wide ESC mutant libraries

GO ID Term Count Fold enrichment Benjamini p value

GO0042254 ribosome biogenesis 29 155 123E-22

GO0022613 ribonucleoprotein complex biogenesis 30 1311 139E-21

GO0034660 ncRNA metabolic process 33 978 457E-20

GO0006396 RNA processing 44 603 275E-19

GO0006364 rRNA processing 22 178 181E-18

GO0016072 rRNA metabolic process 22 1756 207E-18

GO0034470 ncRNA processing 27 1023 824E-17

GO0006260 DNA replication 23 906 691E-13

GO0006412 translation 31 582 101E-12

GO0006259 DNA metabolic process 29 412 307E-08

GO0006261 DNA-dependent DNA replication 10 1761 218E-07

GO0007049 cell cycle 29 284 729E-05

GO0008380 RNA splicing 16 476 809E-05

GO0006397 mRNA processing 18 411 103E-04

GO0006399 tRNA metabolic process 12 636 146E-04

GO0016071 mRNA metabolic process 18 357 600E-04

GO0006270 DNA replication initiation 5 2993 672E-04

GO0031163 metallo-sulfur cluster assembly 5 2993 672E-04

GO0016226 iron-sulfur cluster assembly 5 2993 672E-04

GO0006413 translational initiation 7 1103 154E-03

GO0032774 RNA biosynthetic process 11 523 197E-03

GO0006457 protein folding 11 518 201E-03

GO0000278 mitotic cell cycle 14 343 846E-03

GO0030163 protein catabolic process 22 237 142E-02

GO0009057 macromolecule catabolic process 24 22 195E-02

GO0006268 DNA unwinding during replication 4 2177 221E-02

GO0032508 DNA duplex unwinding 4 1995 279E-02

GO0022402 cell cycle process 17 259 270E-02

GO0006418 tRNA aminoacylation for protein translation 6 781 277E-02

GO0043038 amino acid activation 6 781 277E-02

GO0043039 tRNA aminoacylation 6 781 277E-02

GO0006351 transcription DNA-dependent 9 442 273E-02

GO0032392 DNA geometric change 4 1842 308E-02

GO0022403 cell cycle phase 15 274 311E-02

GO0001824 blastocyst development 6 665 494E-02

Nature Biotechnology doi101038nbt2800

Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

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Supplementary Table 6 A summary of gRNA hits in genome-wide screens

GPI screen MMR screen

1 627 264

2 13 7

3 3 0

4 3 0

5 0 0

6 5 1

7 3 1

8 0 3

9 0 0

10 0 0

Total 654 276

Total numbers of gRNA hits from

both ESC libraries

Number of genes with the indicated number of gRNA hits

Nature Biotechnology doi101038nbt2800

Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800

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Supplementary Table 7 Primer sequences (5 gt 3)

Construction of pEF1a-hCas9

bpA-AgeI-BstZ17I U GATCACCGGTATCGATGTGCGGCCGCCGATGATCTAGAGCTCGCTGATCA

L CAGTGTATACTGCAGGTTCTTTCCGCCTCAGAAG

Construction of pPB-LR51-EF1a-puro2ACas9

pBS-MluI-XbaI U GCTCTAGACAATTCGCCCTATAGTGAGTCGT

L CGACGCGTGCTTTTGTTCCCTTTAGTGAGGG

bpA-ClaI-XhoI U CCATCGATTTAATTAACTGTGCCTTCTAGTTGCCAGCCAT

L CCGCTCGAGCCATAGAGCCCACCGCATCCCCAG

Puro-T2A-GFP U1 GATCCAATTGGGCGCGCCCCACCATGACCGAGTACAAGCCCACGGTG

L1 TCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCATGCA

U2 AGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCACTCGAGGAATTCGGATCCATGGTGAGCAAGGGCGAGGAGCTG

L2 AGCCTTAATTAAGCGGCCGCTCACTTGTACAGCTCGTCCATGCCGAG

EF1a U GATCGCTAGCACGCGTGAGGGGCGCGGAAGCCGGGGCGGAG

L GATCCCATGGTGGTCGACCAAGCTTCCACGACACCTGAAATGGAAGAAAAAAAC

GFP U GATCAAGCTTGGCGCGCCATGGTGAGCAAGGGCGAGGA

L TACGGGATCCTTACTTGTACAGCTCGTCCA

Construction of pKLV-U6gRNA(BbsI)-PGKpuro2AGFP

pBS-MCS-KLV U GATCGGTACCTAGCGGCCGCTGGATCCGGCGCGCCCTCGAGTAACGCGTAGGGCCCATGACTAGTCAATTCGCCCTATAGTGAGTCGT

L GATCCGGCCGTTAATTAAGCTTTTGTTCCCTTTAGTGAGGG

PGKpuro2A U GATCGGATCCAATTCTACCGGGTAGGGGAG

L GCATGTTAGGAGACTTCCTCTGCCCTCTCCTCCGGACCCGCCGCCGGCACCGGGCTTGCGGGTCAT

2ABFP U1 GAGGAAGTCTCCTAACATGCGGTGACGTGGAGGAGAATCCTGGCCCAATGAGCGAGCTGATTAAGGAGA

L1 ACATGCGGCCGCTCAATTAAGCTTGTGCCCCAG

U2 CACCACATACGAGGACGGGGGCGTGCTGAC

L2 GTCAGCACGCCCCCGTCCTCGTATGTGGTG

Mouse whole genome gRNA library construction

79mer U1 GCAGATGGCTCTTTGTCCTA

L1 GCGACGAGAAGACTGTAAAAC

PCR amplification of gRNAs from libraries or enriched mutants for NextGen sequencing

gLibrary-MiSeq_150bp-PE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGAAAGTATTTCGATTTCTTGG

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTACTCGGTGCCACTTTTTCAA

gLibrary-HiSeq_50bp-SE U1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA

L1 TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCATGCTCCAGAC

cDNA cloning

B4galt7 U1 GATCGTCGACCACCATGTTGCCCTCCCGGAGGAAAGCG

L1 GATCTGTACATCAGCCAAATATGCACCATGGGGTG

Ext2 U1 GATCGTCGACCACCATGTGTGCGTCAGTCAAGTCCAA

L1 GATCGAATTCTCATAAGCTGCCAATGTTGGGGAA

PighD12 and Pighwt U1 TCGTGGGAAGCTTGGTCGACCACCATGGAGGACGAGA

U2 CACCATCTTAGGCCTGCTTGGTTACC

L1 CAGGCCTAAGATGGTGGCAGAGAGGAC

L2 GGAATTGGATCCACAGAATTCATAAGCTTGTTGCTGTG

Nature Biotechnology doi101038nbt2800


UALR Biosciences Core Facility High Throughput DNA Sequencing Fragment Analyses Real Time PCR In situ Hybridization Proteomics.

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