SR01: Advances in CRISPR-Cas9 Tools and Applications for ... · PTC prematur termination codon)...

59th Annual Meeting & ToxExpoMarch 15–19, 2020 • Anaheim, California

SR01: Advances in CRISPR-Cas9 Tools and Applications for Toxicologists

Continuing Education CourseSunday, March 15 | 7:00 AM to 7:45 AM

Chair(s) Cheryl Rockwell, Michigan State UniversityElena Demireva, Michigan State University

Primary EndorserContinuing Education Committee

Other Endorser(s)Mechanisms Specialty Section

Molecular and Systems Biology Specialty Section

Presenters Elena Demireva, Michigan State University

Christopher Vulpe, University of Florida

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7:05 AM–7:25 AM Latest Advances in CRISPR-Cas Technologies Elena Demireva, Michigan State University, East Lansing, MI 4

7:25 AM–7:45 AM Use of CRISPR/Cas9-Based Genome-Wide Screens in Toxicology from a User’s Perspective Christopher Vulpe, University of Florida, Gainesville, FL 28

Advances in CRISPR-Cas9 Tools and Applications for Toxicologists



Elena DemirevaMichigan State University

East Lansing, MI517.884.6955

Email: [email protected]

Latest Advances in CRISPR-Cas Technologies

Conflict of Interest Statement

The author declares no conflicts of interest including any financial interests or affiliation with any commercial organization that has a direct or indirect interest in the subject matter of this presentation.

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Presenter 1 | SR01



Abbreviations • Acr: anti-CRISPR

• BE, ABE, CBE: base editor, adenosine BE, cytosine BE

• BER: base excision repair

• Cas: CRISPR-associated

• CRISPR: clustered regularly interspaced short palindromic repeats

• CRISPRa: CRISPR activation

• CRISPRi: CRISPR interference

• crRNA: CRISPR-related RNA

• dCas9: ‘dead’/catalytically inactive Cas9

• DSB: double strand break

• ESC: embryonic stem cells

• GE: genome editing

• HA: homology arm

• HDR: homology directed repair

• Indels: insertions and deletions

• MMEJ: microhomology-mediated end joining

• MMR: mismatch repair• NHEJ: non-homologous end joining• PAM: protospacer adjacent motif• NTS: non-target strand• PE: prime editor• PFS: protospacer flanking sequence• PTC: premature termination codon• PRA: recombinase polymerase

amplification

• RNAP: RNA Polymerase• sgRNA (gRNA): single guide RNA• tracrRNA: transactivating crRNA • Seq: sequence/sequencing • SHERLOCK: Specific High-Sensitivity

Enzymatic Reporter UnLOCKing• SSA: single strand annealing• SSTR: single stranded template

repair• ssODN: single stranded

oligodeoxynucleotide• TS: target strand

Course Outline

1. Background on CRISPR in bacteria and eukaryotic cells

2. From SpCas9 to an expanded CRISPR universe

3. Diversity, evolution, and engineering of CRISPR systems

4. Cas Effectors beyond programmable nucleases

5. Rapidly evolving CRISPR-Cas applications—from Base Editing to RNA Targeting

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In Bacteria: CRISPR-Cas Provides Adaptive Immunity

In nature, bacteria adapt to invading viruses by acquiring immunity to foreign genetic elements to protect against subsequent infections by the same pathogen

I. Adaptation II. Expression III. Interference and Immunity

Cas proteins CRISPR array

Bacterial cell

Viral DNA

Detection of foreign DNA

Cas1-Cas2

Transcription

Cas

Effector nuclease

Surveillance complex Immunity

Processing & assembly

Integration

Target interference

crRNAs

Knott and Doudna, 2018

In Eukaryotic Cells: Streptococcus pyogenes Cas9 First Adapted for DNA Editing

Jinek et al., 2012 ScienceJinek et al., 2013 ElifeCong et al., 2013 Science

crRNA

tracrRNAcrRNA + tracrRNA complex

single chimeric RNA = sgRNA (gRNA)

sgRNA

GCUGGAACUUAUGCAAC GCU GUUUUAGAGCUA

GCCUAGAUCGGAAUAAAAUU CGAU. .

GAA

GAAA

5’ -

3’ -

scaffold Spacer (guide seq)

5’ -

3’ - target strand (TS)

non-target strand (NTS)

Protospacer (target seq)

sgRNA

genomic DNATarget genomic DNA

sgRNA

SpCas9 PAM

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CRISPR Genome Modifications Result from DNA Repair of DSBs

Ran et al., 2013 Nat Protoc

Error prone but high efficiency.Present in somatic and dividing cells.

Low efficiency, requires a DNA template. Limited in non-dividing cells.

DSB

Indel/random mutation

Precise modification

NHEJMMEJ

Rad51-dependentre-ligation of ends

HDRSSTRRad51-independent

Requires template

5’ -3’ - - 5’

- 3’

5’ -3’ - - 5’

- 3’Indel mutation PTC

Indel results in a frameshift and downstream PTC (premature termination codon)

DSB, bound by NHEJ repair machinery

Genomic DNA

DSB, bound by HDR repair machinery

Repairtemplate

Precise modification

5’-3’- -5’

-3’

5’-3’- -5’

-3’

5’-3’- -5’

-3’

Homologous recombination

Precise Genomic Modifications Directed by Different DNA Repair Templates

Template Repair Pathway Properties/LimitationsPlasmid, HA > 500bpdsDNA fragment

HDR • Low efficiency• Long homology arms—large constructs

dsDNA fragments with short HA 30–100bp

MMEJ/SSA • Indels at insertion sites• Off-target integration• May be promising with shorter HAs 24–48bp

ssODN (oligo)Max 200bp, HA 30–40bp

HDR/SSTR

• Limitations of insert size• Reduced off-target/random integration• Both NTS and TS templates can be used• Asymmetry of HA can be considered• Less toxicity than dsDNA templates

Long ss DNA (200bp–3kb)HA 40–200bp

Donor HDR Efficiency FactorsPosition of edit with respect to PAM and seed region of RNA/DNA hybridInsert length and sequence (secondary structures of long ssDNA can result in mutations)Locus dependency—open versus closed chromatin, structural topology, repetitive DNA, empirical testsDistance from DSB (insertion site) to start of homology armsgRNA cutting efficiency—important to validate gRNA efficiency prior to introduction of donor template

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Cas9n

NTS

NTS

TS

TS

• Nicks are repaired with much higher fidelity than DSBs in mammalian cells via the BER pathway

• Mutating the catalytic residue of either nuclease domain of Cas9 converts it to a nickase- Cas9 H840A (HNH domain) nicks non-target strand (PAM strand)- Cas9 D10A (RuvC domain) nicks target strand Jinek et al., 2012 Science

Cong et al., 2013 Science

A Single Mutation Converts SpCas9 to a Nickase

Double nicking by Cas9n (D10A) with paired gRNAs

Ran et al., 2013 Cell

• Cas9n with paired gRNAs on opposite strands introduces DSBs with high specificity.

• Off-target activity reduced by 50- to 1,500-fold

• DSBs induced by double nicking are effectively repaired by both NHEJ and HDR

• Distance between nicks can be optimized

• HDR facilitated when gRNA orientation results in a 5’ overhang

Faster and cheaper (weeks to months to generate edited models)Easily accessible and adapted for different applicationsInherently programmable and easy to engineer, unlike ZFNs and TALENs editors (RNA-based versus protein-based recognition of target DNA)Functional in many species: mammalian, insects, nematodes, fish, and plants

Amenable for in vivo and ex vivo deliveryDirect editing of vertebrate and invertebrate embryos from multiple speciesDifferent formats for delivery—plasmid, RNP, virus, etc.Allows for multiplexing (generate multiple genomic modifications simultaneously)

No “scarring” at genome modification site

Applications beyond DSB generation, such as transcriptional regulation, genome imaging, epigenetic modifications, molecular recoding, chromatin looping

Advantages of CRISPR-Cas over Other GE Methods

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SpCas9 Research Has Paved the Way for Next-Gen Genome Engineering

• Extensively characterized for predicting on-target efficiency and off-target effects, tested and adopted for many different species and systems, new applications for whole genome and targeted screens, in vivo editing, lineage tracing, data recording using DNA, gene drives, and more.

• Major tool for genome editing in last seven years. Superior to prior methods such as ZFN/TALEN, ESCs HDR, transgenesis, siRNA.

• Optimization of repair template formats (ssODN, long ssDNA, dsDNA) for more efficient precise editing. Understanding of DSB DNA repair mechanisms.

• Vast natural diversity of CRISPR homologs and availability of engineered variants.• Structural studies of Cas9 complexes and extensive insertional mutagenesis studies—

inform how to engineer multi-protein modular Cas effector platforms for diverse applications.

SpCas9 DNA Editing Has Led to an Explosion of New Discoveries and Advances in the GE Field

• Natural diversity and evolution of CRISPR-Cas systems

• Engineering Cas Effectors beyond nuclease activity

• Restricting CRISPR-Cas activity with Anti-CRISPRs

• RNA editing with Cas13 and related applications

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Naturally Evolved Diversity of CRISPR SystemsClass 1 Class 2

Koonin et al., 2017 Curr Opin Microbiol

Mine Cas protein orthologs for new and distinct properties: PAM flexibility, smaller size, naturally high specificity, pre-crRNA processing activity, DNA shredders, RNA targeting

Exploring the Natural Diversity of CRISPR Systems

• Remarkable diversity across species of bacteria and archea: protein components, effector complex, cas operon locus architecture, and pre-crRNA processing.

• Class 1—multi-protein effector complexes

• Class 2—a single-component effector protein (e.g., Cas9 [Class 2, Type II subtype])

• All CRISPR-Cas systems rely on crRNA for guidance and targeting specificity

• The adaptation module, composed of Cas1 and Cas2 endonucleases, is shared by all known CRISPR-Cas systems

• Diversity observed at the level of processing of the pre-crRNA to mature crRNA guides, either via a Cas6-related ribonuclease or a housekeeping bacterial Rnase III

• More recently characterized Class 2 Type VI systems are the first variants to exclusively target RNA

Koonin et al., 2017 Curr Opin Microbiol

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Properties of Cas9 Orthologs and Engineered Variants

Cas9 protein Protein variant or mutations PAM (5′ to 3′) Properties/SizeSpCas9 Native Streptococcus pyogenes Cas9 NGG 1,368 amino acids

xCas9-3.7A262T, R324L, S409I, E480K, E543D, M694I, E1219V NG, GAA, GAT Altered PAM variant

eSpCas9 (1.1) K810A, K1003A, R1060A NGG Enhanced specificityCas9-HF1 N497A, R661A, Q695A, Q926A NGG Enhanced specificityHypaCas9 N692A, M694A, Q695A, H698A NGG Enhanced specificity

HiFi Cas9 R691A NGGEnhanced specificity for RNP

ScCas9 Native Streptococcus canis Cas9 NNG 1,375 amino acidsStCas9 Native Streptococcus thermophilus Cas9 NNAGAAW 1,121 amino acidsNmCas9 Native Neisseria meningitidis Cas9 NNNNGATT 1,082 amino acidsSaCas9 Native Staphylococcus aureus Cas9 NNGRRT 1,053 amino acidsCjCas9 Native Campylobacter jejuni Cas9 NNNVRYM 984 amino acidsCasX Phyla Deltaproteobacteria and Planctomycetes TTCN 980 amino acids

Cas9 variants with alternate protospacer adjacent motif (PAM), targeting specificities, and protein size

Pickar-Oliver and Gersbach 2019 Legend: N = any base, M=A/C, R=A/G, W=A/T, V= A/C/G, Y=C/T, Engineered, Naturally occurring

crRNA

pre-crRNA

5’- 5’-5’-

Processing of pre-crRNA by Cas12a’s ribonuclease activity

Cas12a

Gene 1 Gene 2 Gene 3

Multiplex Editing

Cas12a (Cpf1)—A Class II, Type V RNA-Guided Endonuclease

• Exhibits robust DNA interference, distinct features from Cas9

• Single crRNA guide, no tracrRNA

• T-rich PAM (5’-TTN)

• Staggered DSB results in 5’ overhangs

• Intrinsic ribonuclease activity used for processing of pre-crRNA

• Single pre-crRNA allows for multiplexing

• 16 Cas12 family members with diverse properties

Zetsche et al., 2015 CellZetsche et al., 2017 Nat Biotech

|GGTACCCGGGGATCCTTTA GAGAAGTCAT TTAATAAGGCCACTGTTAAAAAGCTTGGCGTAATCA

CCATGGGCCCCTAGGAAATCTCTTCAGTAAA TTATTCCGGTGACAATTTTTCGAACCGCATTAGT

UGUAGAUGAGAAGUCAUUUAAUAAGGCCACU

UCAUCUUUAA

| ||| | |||| ||| | ||| ||| | ||| | || ||| | || |||

| ||| || || |||| ||| ||

| | |

protospacer

|| | |

PAM

||| | | |

UG

|

3’ - ···

5’- ···

··· - 5’

··· - 3’

- 5’

- 3’

| |

|

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Dolan et al., 2019 Mol Cell

cas3 cse1 cas5ecas7

CRISPR arraycas6ecse2 cas1 cas2

8+32+21=61bpcrRNA

spacer acquisition

Cascade complexgRNA target recognition

target degradation

Class I CRISPR Systems—Cas3 and Cascade• Most abundant in nature (~90% of CRISPR systems)• A multimeric DNA-targeting complex (Cascade) and a Cas3 helicase-nuclease • Cascade consists for 8-11proteins—multiple options for attachment of accessory modules• crRNA recognition spans >30bp providing potential for high specificity in larger genomes• Promiscuous PAM offers greater targeting flexibility • Cas3 nicks DNA upon recruitment and degrades target DNA upstream of PAM via 3’ -> 5’

exonuclease activity• CasE, a ribonuclease, mediates guide RNA processing—transcription of several guides from

the same promoter

Class I CRISPR Systems—Cas3 and Cascade• Cas3 exhibits great processivity of ssDNA degradation (DNA shredding)—allows for

long-range chromosomal modifications in human cells

• Many potential applications—antimicrobial, removal of transposons/integrated viral elements, exon-skipping, screening of non-coding regions

Dolan et al., 2019 Mol Cell

Deletion mapping in human ESCs

A spectrum of deletions(0.5–100kb) are generated

unidirectionally from target site

-50kb -20kb -5kb-10kb

Translocation direction

genomic DNACascade

PAM

D onset range ~400bp

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Cas Effectors beyond Nucleases

• Harness dCas9-specific DNA binding and processivity to recruit other functional domains/modules to precise genomic loci

• Avoid DSB toxicity and unwanted editing by-products• Cas-effector platforms offer limitless potential functionalities• Can be designed with both Class 1 and Class 2 CRISPR systems

1. Transcriptional regulators—CRISPRa and CRISPRi2. Epigenetic modifiers—alter DNA methylation and histone modification3. Base Editors—change single nucleotides without causing DSB4. Prime Editing—precision modification without DSB and need for a repair

template5. Other applications—chromosome imaging, identifying chromatin interactions

dCas9 linked to effector domains can potentially recruit any protein to any DNA sequence

Gilbert et al., 2013 Cell

Cas Effector Platforms

Genomic DNA target• Coding DNA• Non-coding DNA• Promoters• Regulatory elements• CpG island

DNA Targeting

dCas9

Effector Transcription activator/repressorEpigenetic modifier: CpG-Me, His-Ac, His-Me

Base Editor: A>G, C>TFluorophore, epitope—imaging, ChiP

Target-Gene or Element

Regulation

Effectors:• Proximal—edit DNA within target region• Distal—act on DNA adjacent to target • Fusions, linker-mediated, multi-modular,

accessory factor recruitment

gRNA

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• CRISPR activation—dCas9 is fused to transcriptional activator domains in a multi-modular cooperative manner to activate gene expression when guided to promoter regions

• Several different systems exist, including VPR, SAM, and SunTag activation systems

Transcriptional Effectors: CRISPRa

mRNA

SunTag

VP64

scFV-epitope recruitment

MS2p65HSF1

VP64

SAM

mRNA

VP64p65

RtamRNA

VPR

dCas9

Tanenbaum et al., 2014 CellKonermann et al., 2015 NatureChavez et al., 2015 Nat Methods

Tycko et al., 2017 Nat Methods

Transcriptional Effectors: CRISPRiCRISPR interference—repression of gene expression, two main approaches:1. Steric hinderance between dCas9 complex and transcriptional machinery,

depending on location can block transcription initiation or block elongation when RNAP collides with dCas9

2. dCas9 is fused to transcriptional repressor domains

Qi et al., 2013 Cell Larson et al., 2013 Nat Protoc

dCas9RNA Pol

Direction of transcriptionCollision with dCas9 complex

target locus

gRNA

dCas9 KRAB

Co-repressors

Gilbert et al., 2013 Cell

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Epigenetic Effectors: Histone Modification• dCas9 epigenetic effectors can write or erase histone modifications, or directly alter DNA methylation

• Epigenetic effectors can be used to transiently or stably activate or repress specific genes or annotate non-coding genomic regions by changing heterochromatin/chromatin status

• Chromatin modifiers change acetylation or methylation of histones: - Acetylation modified by p300 HAT and HDAC3 effectors - Methylation modified by LSD1, KRAB, PRDM9

Hilton et al., 2015 Cano-Rodriguez et al., 2016Kearns et al., 2015 Kwon et al., 2017Gilbert et al., 2013Histone Modification

MeMe

target locus

dCas9

Histone modifying enzyme

gRNA AcAc

P300HDAC3

PRDM9 LSD1, KRAB

Epigenetic Effectors: DNA MethylationdCas9 epigenetic effectors that are coupled to DNA methylation enzymes include:

- Fusion of dCas9 to Tet1 for erasing methylation of CpGs- Fusion to Dnmta3 for de novo methylation of specific sequences

Liu et al., 2016 Cell

target locus

dCas9

gRNAC

Me

CMe

CMe

Dnmt

target locus

gRNACCC

TetdCas9

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Improved Precision Editing Cas Effectors: Base EditorsTo achieve efficient editing of human pathogenic point mutations:• Increase precise editing efficiency over that of Cas9+HDR (typically 0.1%–5%), especially in somatic

cells• Reduce random editing products of NHEJ repair (indels); e.g., bi-allelic edits, mixture of genotypes• Eliminate potential DSB toxicity due to random editing by-products, unintended structural changes

such as chromosomal rearrangements, and activation of p53 pathway• Improve in vivo delivery of editing systems: eliminate co-delivery of DNA repair template

To address these goals, Dr. David Liu’s group has developed a series of base editors (BE):ABE—adenine base editorsCBE—cytosine base editors

• Allow for all four transition mutations (C→T, G→A, A→G, and T→C) • Expand the type of edits that can be achieved cleanly• Improve overall SNP repair efficiency• Expand the type of cells that can be targeted with BEs

CBE: Change a Single or a Window of NT from C>T (A>G)

• dCas9 or Cas9 nickase (nCas9) is fused to cytidine deaminases, which is a ssDNA deaminase targeting C residues in the Cas9 R-loop

• Deaminases combined with Cas9 nickase and uracil DNA glycosylase inhibitor modules increase the efficiency of changing cytidine to thymidine

• Repair T>C mutations or create KO or inactivate dominant negative alleles via the iSTOP approach

• iSTOP—convert any of four codons (CAA, CAG, CGA, and TGG) into a termination codon

Komor et al., 2016 Nature Kim et al., 2017 Nat BiotechnolBillon et al., 2017 Mol CellTycko et al., 2017 Nat Meth

*C G C

T C G

Arg C G A

T G A

Arg

STOP

Precise SNP repair

Ser

Introduction of STOP codon

NickaseCas9

Cytidine deaminase

Uracil DNAglycosylaseinhibitor

C>T

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CBE: Mechanism of Action• Deamination of cytosine (C) by a cytidine deaminase domain results in uracil (U) conversion• U can base-pair as a thymine (T), and upon DNA mismatch repair or replication replaced by a T• Selected a cytosine deaminase that uses ssDNA as a substrate—has access to the first 11bp of protospacer• First-generation BE can convert C to T within a short window of position 4–8 of protospacer (distal to PAM)

Komor et al. 2016 Nature

Cytidine deaminase

NickaseCas9

G

C

G

UPAMProtospacer

GC

AT

Deamination of target C

DNA replication or repair

gDNA binding and opening

Genomic DNA

Base-edited DNA

Editing window

5’ ···

3’ ··· ··· 5’

··· 3’

3’5’

5’3’

CBE Engineering—Higher Efficiency, Flexible PAM, Narrow Editing Window

Komor et al., 2016 Nature

C G G C G

C G

U A

U G

U G

T A

Cytosinedeamination

Blocked by BE2 and BE3

Cellular base-excision repair

DNA replication or repairCellular

mismatch repair

BE1, BE2, or BE3

Cellular uracil DNA glycosylase

BE3 favors repair of nicked strand

Nicking by BE3

Desired base edit

BE3 disfavors repair of intact strand

BE1: rAPOBEC1-XTEN-dCas9BE2: rAPOBEC1-XTEN-dCas9-UGI BE3: rAPOBEC1-XTEN-Cas9n-UGI-NLS

Further evolve variants to expand PAM range and reduce editing window Base editor PAM BE3 NGG VQR-BE3 NGAN EQR-BE3 NGAG VRER-BE3 NGCG SaBE3 NNGRRT SaKKH-BE3 NNNRRT

Approx. Editing Window (nt)Base editor Site A Site BBE3 4 6YE1-BE3 2 2YE2-BE3 2 2EE-BE3 2 2YEE-BE3 2 1

0%10%20%30%40%

Cas9 +ssODN

BE1 BE2 BE3

Relative BE efficiencies

% E

dite

d Ta

rget

Cs

Rees et al., 2017 Nat Comm

1. UGI (uracil glycosylse inhibitor) added to BE2 and BE32. Nickase (Cas9n) function restored in BE3

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DNA deoxyadenosine deaminase

NickaseCas9

T

A

T

I

DNA replication or repair

gDNA binding and unwinding

CG

Base-edited DNA

PAMProtospacerA

Genomic DNAT

Deaminate A in ssDNA bubble

Nick non-edited strand5’ ···

3’ ··· ··· 5’

··· 3’

3’5’

··· 5’

3’5’

··· 3’

Gaudelli et al., 2017 Nature

ABE: Engineering of A>G (T>C) Editors• Engineer a novel DNA deoxyadenosine deaminase from transfer RNA adenosine deaminase by

directed evolution mutagenesis, and fuse to a Cas9 nickase• Deaminate adenosine (A) to convert to inosine (I), which can base-pair with C and be replaced with G

after DNA repair or replication• Simultaneous nicking of non-edited strand favors removal of T instead from mismatch• Engineered seventh-generation ABEs have 50% editing efficiency in human cells, and <0.1% indel rate

• ABEmax and CBEmax with expanded targeting scope: e.g., xABEMax = ecTadA-ecTadA*(7.10)-nSpCas9 (xCas9)CP-CBEMax = rApobec1-nSpCas9 (CP#) -UGI-UGI

Huang et al., 2019 Nat Biotechnol 37(6):626

• Continuous directed evolution of base editors to improve editing efficiency and target sequence compatibility

Thuronyi et al., 2019 Nat Biotechnol 37(9):1070

Newest Base Editor Variants

• BE4Max, evoAPOBEC1-BE4max, evoFERNY -BE4max, evoCDA1-BE4max, and more . . .

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The Latest from Dr. David Liu’s Laboratory:

Anzalone et al., Nature 2019

Prime Editing• A new method for introduction of small targeted insertions or deletions, and

base conversions without DSB, or donor template • Possible edits with PE:

- All four transition point mutations (C>T, G>A, A>G, T>C), - All eight transversion point mutations (C>A, C>G, G>C, G>T, A>C, A>T, T>A, T>G) - Insertions (1 bp to ≥ 44 bp) - Deletions (1 bp to ≥ 80 bp)

• Higher or similar efficiency and fewer by-products than DSB/HDR-based CRISPR• Additional DNA-RNA hybridization points in PE greatly decrease off-target effects

Overview of Prime Editing

Primer editor and pegRNA

Cas9 nickase

pegRNA3’-

5’-

Reverse transcriptase (RT) domain

PAM

Protospacer

pegRNA nick site Primer Editing rangefor Cas9 PE

5’ ···

3’ ···

··· 3’

··· 5’

Target DNA • Cas9 nickase fused to an engineered M-MLV reverse transcriptase (RT)

• A 3’ extended prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit

• pegRNA binds to primer binding site (PBS) on the edited strand

• Hybridization of pegRNA to primer binding site primes RT to direct synthesis of the edited DNA

• 5′ flap excision and 3′ flap ligation from a branched intermediate drive incorporation of the edited DNA strand creating heteroduplex DNA

• DNA repair resolves the mismatched heteroduplex to copy the edit on the complementary strand


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Primer 8–15ntGGCCCAGACTGAGCACG TGA NGG CAGAC

nickDNA to be

replaced, ≥ 7bpEdited strand of target DNA

5’ ··· ··· 3’option to edit PAM

5’ – GGCCAGACUGAGCAGCUGAAGUUUUAGAGCUA· | | | | | | · | | | |

C-GGAAUAAAAUU CGAU| | | | · | G AGUCCGUUA

UC

AACUU· | | | |

GCCACGGUAA| | | | | |

CGGUGCUCUGCCAUCUCGUGCUCAGUCUGUUU– 3’

spacer/guide seq

RT templatewith edit

Primer-binding site

U

AG

A

AA

A

A

G

G

AA

U

pegRNA

Assembly of PE + pegRNA and genomic DNA

Nicking of PAM Strand

3’-

Cas9 nickase

pegRNA

Reverse transcriptase (RT) domain



RT template containing editPrimer-binding site

Reverse transcription

Hybridization of primer-binding site in pegRNA to PAM strand

Direct polymerization of edited DNA from pegRNA into target site

Flap equilibration

3’ flap (with edit)

5’ flap (no edit) 5’ flap

cleavage

Ligation

DNArepair Edited DNA

Branched intermediates with two redundant ssDNA flaps

Spacer/guide sequence



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• Both BE and PE allow for precise, targeted mutations without inducing DSBs, requiring DNA donor templates, or relying on low-efficiency HDR pathways

• Both can edit human cells and work in dividing and somatic cells

• Both have a reduced number of components that need to be delivered for in vivo and therapeutic applications

• BEs are useful for single nucleotide transition mutations, with newer BE versions performing with higher efficiency and almost undetectable indel rates

• PEs are less dependent on PAM location or local sequence constraints

• PEs can generate targeted deletions, insertions, and all possible 12 single-base conversions

• PE can theoretically correct the majority (~89%) of all known human pathogenic genetic variants

• PEs have higher indel rates than BE; therefore, for transition mutations PE or BE should be used depending on the desired edit and the target sequence context

Summary of Base Editing (BE) and Prime Editing (PE) Technologies

Restricting CRISPR-Cas Activity with Anti-CRISPRs• Anti-CRISPRs (Acr) are small protein inhibitors of CRISPR systems (~90 a.a)• Acrs evolved in bacteriophage as defenses against CRISPR immune

systems of their bacterial host • Function as “off switches” of Cas9 activity in mammalian cells• Many applications for effective control of Cas9 activity:

• Restrict Cas9 expression temporally or spatially• Prolonged Cas9 expression increases off-target effects and risk of

chromosomal abnormalities—especially problematic for therapeutic applications

• In development of GE models, prolonged Cas9 activity can lead to mosaicism of edited alleles and increase frequency of random indels

• Potential fail-safe measure to prevent gene-drive propagation • Robust, versatile, and genetically encodable approach to limit Cas9

activity compared with other methods, such as drug-inducible or light-inducible Cas9 variants

Pawluk et al., 2016 Cell

Liu et al. 2019 Mol Cell 73(3):611

In vitro enzymatic assay of WT AcrIIA2 and AcrIIA2 with Ala substitutions of residues that interact with sgRNA-bound SpyCas9

1—DNA substrate2—DNA + SpCas9-sgRNA 3—DNA + SpCas9-sgRNA + WT ArcrIIA24—111 DNA + SpCas9-sgRNA + Mut ArcrIIA2

1 2 3 4 5 6 7 8 9 10 11

Liu et al., 2019 Mol Cell

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Anti-CRISPRs• Encoded by acr genes, which parallel the diversity of CRISPR

systems• Acrs evolved against both class 1 (AcrI) and class 2 (AcrII)

systems • Distinct Acr families inhibit CRISRP-Cas of different subtypes

and species—providing options for narrow and broad inhibition

• Known mechanisms of how Acrs block DNA cleavage by Cas9: - Disabling nuclease domains without effect on DNA binding- Blockade of catalytic DNA cleavage site- Dimerization of Cas9 to prevent active conformation- Destabilization dCas9 binding to DNA by obstructing PAM access

• Versatility of Acr mechanisms offers flexibility in designing Cas9 inhibition strategies for different applications

Pawluk et al., 2016 Cell Liu et al., 2019 Mol Cell

A. Domain organization of SpyCas9.

B. Ribbon and surface representations of the AcrIIA2-SpyCas9-sgRNA complex. The color-coding used for Cas9 is identical to that used in (A). AcrIIA2 is shown in magenta.

Harrington et al., 2017 Cell

A

B

(4) RNA-Targeting CRISPR-Cas Systems• Cas13 (Class 2, Type VI) systems are programmable RNA-guided RNases • Cas13 family contains at least four subtypes (Cas13a, Cas13b, Cas13c, and Cas13d)• Evolved as defense against RNA bacteriophages or to degrade transcripts of DNA viruses• Collateral RNase activity after cleavage of target transcript leads to nonspecific RNA degradation of

nearby transcripts (undetectable in mammalian or plant cells) • Expression of Cas13 proteins with crRNA and target RNA leads to cellular toxicity, suggestive of

induction of dormancy or programmed cell death in bacteria• Main advantage—changes to gene expression or function can be transient and reversible and

engineered without a permanent disruption of genome• RNA-targeting is not reliant on cellular DNA repair pathways• Applications:

• Transcript knockdown therapeutics, RNA base editing, antimicrobial, alternative splicing• CRISPR-Dx—rapid, cheap, portable, and ultrasensitive detection of nucleic acids

Abudayyeh et al., 2016 Science Pickar-Oliver and Gersbach, 2019 Nat Rev Mol Cell Biol

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RNA Targeting by Cas13

Mature crRNA

Generalized Type VI locus crRNA processing

CRISPR arraycas13 cas1 cas2

5’-

viral/target RNA

mRNA

Cas13

Degradation of viral/target ssRNA

Nonspecific (collateral)degradation of nearby RNA

Phage

Abudayyeh et al., 2016 Science

Target specificity encoded by a 28–30nt spacer of crRNA

Cas13 complexes with crRNA via hairpin recognition

Diagnostic Applications of Cas13 (SHERLOCK)• Co-opt nonspecific RNase activity to cleave fluorescent RNA reporters upon target recognition

allowing real-time detection of the target and combine with isothermal amplification (RPA)• Method achieves ultrasensitive (aM) detection of nucleic acids with 1bp sensitivity

- Single molecule detection confirmed by ddPCR• Many applications—pathogen detection, rapid genotyping, identification of tumor mutations,

environmental applications, epidemiology

Gootenberg et al., 2017 Science

Collateral cleavage of reporter releases signal

Cas13a detection

T7 transcription

RPA

RT-RPA

RNA

dsDNA

cleavage reporter(quenched fluorescent RNA)

Cas13a-crRNA target sequence

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Summary and ConclusionsRapid adoption and success of SpCas9 editing in eukaryotic systems led to “CRISPR revolution”• Propelled discovery of multiple new systems to target DNA and RNA, manipulate epigenome and transcriptome• Led to development of new genome engineering applications—research, diagnostic, translational, environmental• Development of Cas effector platforms with potential to recruit any protein unit to any DNA/RNA sequence

Diversity of CRISPR Cas systems• Addresses PAM flexibility, Cas protein size, specificity, component requirements, distinct nuclease, Cascade + Cas3

new tools for mammalian cell GE • Diversity of CRISPR and anti-CRISPRs offers vast expansion of CRISPR tools for existing and new applications• Directed evolution and protein engineering—key to creating tailored CRISPR systems with novel functionality

Next-gen precise GE • New and rapidly evolving Base Editing and Prime Editing• Precise changes in DNA or RNA, without DSB, need for donor template, or reliance on HDR repair pathways • Important advance for clinical applications, in vivo delivery, and editing of somatic cells

Thank You for Your Attention!

Acknowledgements:

Support:Michigan State UniversityOffice of the SVP for Research and InnovationInstitute for Quantitative Health Science and Engineering

Michigan State University:Dr. Huirong XieBana AbolibdehDr. Richard Neubig

SOT OrganizersDr. Cheryl RockwellDr. Christopher VulpeKevin Merritt

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• Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, SemenovaE, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353(6299):aaf5573.

• Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature576(7785):149–157.

• Billon P, Bryant EE, Joseph SA, Nambiar TS, Hayward SB, Rothstein R, Ciccia A. (2017). CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons. Mol Cell 67(6):1068–1079.

• Cano-Rodriguez D, Gjaltema RA, Jilderda LJ, Jellema P, Dokter-Fokkens J, Ruiters MH, Rots MG. (2016). Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat Commun 7:12284.

• Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-OvanesyanD, Braff JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM. (2015). Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12(4):326–8.

• Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121): 819–823.

• Dolan AE, Hou Z, Xiao Y, Gramelspacher MJ, Heo J, Howden SE, Freddolino PL, Ke A, Zhang Y. (2019). Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas. Mol Cell74(5):936–950.

References

• Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. (2017). Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551(7681):464–471.

• Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, BassikMC, Qi LS, Kampmann M, Weissman JS. (2014). Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell 159(3):647–61.

• Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, FreijeCA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356(6336):438–442.

• Harrington LB, Doxzen KW, Ma E, Liu JJ, Knott GJ, Edraki A, Garcia B, Amrani N, Chen JS, Cofsky JC, Kranzusch PJ, Sontheimer EJ, Davidson AR, Maxwell KL, Doudna JA. (2017). Cell 170(6):1224–1233.

• Hilton IB, D’Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA. (2015). Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol33(5):510–7.

• Huang TP, Zhao KT, Miller SM, Gaudelli NM, Oakes BL, Fellmann C, Savage DF, Liu DR. (2019). Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat Biotechnol 37(6):626–631.

• Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816–821.

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• Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. (2013). RNA-programmed genome editing in human cells. Elife2:e00471.

• Kearns NA, Pham H, Tabak B, Genga RM, Silverstein NJ, Garber M, Maehr R. (2015). Functional annotation of native enhancers with a Cas9-histone demethylase fusion. Nat Methods 12(5):401–403.

• Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR. (2017). Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol. 35(4):371–376.

• Knott GJ, Doudna JA. (2018). CRISPR-Cas guides the future of genetic engineering. Science 361(6405), 866–869.• Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. (2016). Programmable editing of a target base in genomic DNA

without double-stranded DNA cleavage. Nature 533(7603): 420–424.• Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS,

Nishimasu H, Nureki O, Zhang F. (2015). Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517(7536):583–588.

• Koonin EV, Makarova KS, Zhang F. (2017). Diversity, classification and evolution of CRISPR-Cas systems. Curr OpinMicrobiol 37:67–78.

• Kwon DY, Zhao YT, Lamonica JM, Zhou Z. (2017). Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC. Nat Commun 8:15315.

References

• Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc 8(11):2180-2196.

• Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D, Young RA, Jaenisch R. (2016). Editing DNA Methylation in the Mammalian Genome. Cell 167(1):233-247.

• Liu L, Yin M, Wang M, Wang Y. (2019). Phage AcrIIA2 DNA Mimicry: Structural Basis of the CRISPR and Anti-CRISPR Arms Race. Mol Cell 73(3):611-620.

• Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR. (2016). Naturally Occurring Off-Switches for CRISPR-Cas9. Cell 167(7):1829-1838.

• Pickar-Oliver A, Gersbach CA. (2019). The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol 20(8):490-507.

• Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173-83.

• Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6):1380–1389.

• Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. (2103). Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308.

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• Rees HA, Komor AC, Yeh WH, Caetano-Lopes J, Warman M, Edge ASB, Liu DR. (2017). Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun. 8:15790.

• Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD. (2014). A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159(3):635–46.

• Thuronyi BW, Koblan LW, Levy JM, Yeh WH, Zheng C, Newby GA, Wilson C, Bhaumik M, Shubina-Oleinik O, Holt JR, Liu DR. (2019). Continuous evolution of base editors with expanded target compatibility and improved activity. Nat Biotechnol 37(9):1070–1079

• Tycko J, Hess GT, Jeng EE, Dubreuil M, & Bassik MC. (2017). The expanding CRISPR toolbox. Nature Methods Web Poster

• http://s3-service-broker-live-19ea8b98-4d41-4cb4-be4c-d68f4963b7dd.s3.amazonaws.com/uploads/ckeditor/attachments/7742/CRISPR_poster-WEB.pdf

• Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163(3):759–71.

• Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F. (2017). Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol 35(1):31–34.

References

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Use of CRISPR/Cas9-Based Genome-Wide Screens in Toxicology from a User’s Perspective

Christopher VulpeUniversity of Florida

Gainesville, FLPhone: 352.294.5821

Email: [email protected]

Conflict of Interest Statement

The presenter does not have any financial interest or affiliation with a commercial organization that has a direct or indirect interest in the subject matter of my presentation.

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Course Objectives/Outline

1. CRISPR as tool in functional toxicology2. Targeted CRISPR versus genome-wide CRISPR in

toxicology3. Applications of targeted CRISPR in toxicology4. Applications of genome-wide CRISPR in toxicology5. Future applications of CRISPR in toxicology6. CRISPR conclusions and caveats

Abbreviations

CRISPR: clustered regularly interspaced short palindromic repeats

AOP: adverse outcome pathway

sgRNA: single guide RNA

MOI: multiplicity of infection

NGS: next-generation sequencing

KO: knockout

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CRISPR Enables Functional Toxicology

Functional profiling—systematically testing multiple genes for their functional role, if any, in toxicity, by perturbing their function

The study of the requirement for the biological activities of genes and corresponding proteins in the response to, and effect on, an organism by a toxicant

OR if you muck it up (the gene) and bad (or good) things happen, then it’s probably important

Gene Function ToxicityAssess function

in cell or organism

As related to role, if any, in

toxicity

Usually mutate

gene

Targeted CRISPR versus Genome-Wide CRISPR

Gene 1KO

Xone

oneGene of interestone

one

Targeting sgRNA togene of interest CAS9

Introduce intocell/organism

Xone

Screen for KO

Isolate KO cell/organism Assess function

in cell/organism

two three n

Multiple genes of interest

Targeting sgRNA to genes of interest

Xone

1X

two

2X

three

3Xn

n

Introduce into cell One per cell

Gene 1KO

Gene 2KO

Gene 3KO

Gene nKO

Generate pool of mutants

Screen for sensitivity to toxicantto identify the important genes

(CRISPR KO library)

Targ

eted

CRI

SPR

Genome-W

ide CRISPR

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Current Applications of CRISPR in Toxicology

• KO models• Cell lines

• Drug Disposition/Transport/Metabolism• MDR1• P450- CYP3A5 *3 Huh-7

• Mechanistic studies• Nrf2- tBHQ effects attributable to Nrf2• SNAP29—arsenic and autophagy

• Animal models• Ahr2 KO in zebrafish• P450 KOs in rodents—2E1, 3A1/2, 2C11, 2B9/10/13• MDR1

• Reporter Assays• Sox2 reporter• AHR—CYP1A1 reporter—human iPSCs

Single Gene ModificationsGenome-Wide Screening Approaches

Gene 1KO

Assess function/phenotype

in cell/organism

Gene 1KO

Gene 2KO

Gene 3KO

Gene nKO

Pool of mutants

Gene nKO

Identifykey

genes

Toxicant Toxicant

or

Sensitivity

Toxicant and ToxinsAcetaldehydeArsenic Trioxide- Cytotoxicity APAPC. difficile Toxin A & BTriclosanParaquat

Regulators and Response PathwaysAHR InductionNRF2 inductionArsenic—unfolded protein responseUnfolded protein responseKarlgren et al., Drug Metab Dispos 2018;46:1776–1786

Karlgren et al., Drug Metab Dispos 2018;46:1776–1786 Karlgren et al., Journal of Pharmaceutical Sciences 106 (2017) 2909–2913

4 bp deletion in exon 4—leads to FS

Express Human MDR1

KO MDCKCanine MDR1

Characterize multiple individual clones

CRISPR “KO” of Toxicant Transporter in a Cell Line

MDR1

KO canine MDR1 Replace with human MDR1

CRISPR KO must be VERIFIED

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Nrf2 KO to identify Nrf2-specific effects

of oxidants

Single vector-based KO—sgRNA/Cas9 Test several sgRNA

Zagorski JW, Maser TP, Liby KT, Rockwell CE. J Pharmacol Exp Ther. 2017;361(2):259–67.

Individual Clone characterization

Induction of NQO1 is Nrf2 dependent

Suppression of IL-2Not dependent on Nrf2

Targeted CRISPR KO to Assess Mechanism of Action of Toxicants

Garcia GR, Bugel SM, Truong L, Spagnoli S, Tanguay RL (2018) PLOS ONE 13(30)

New Animal Models: Zebrafish KO of AHR2

NLS

Form an in vitro ribonucleoprotein complex

Direct injection—1 cell embryo

Target exon 1 of Zf AHR2

Screen for germ line transmission—2–4 monthsEstablish founder lines

Screen for KO

AHR2 truncation

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Ahr2 KO Mutants Are Resistant to TCDD-Induced Developmental Toxicity

Garcia GR, Bugel SM, Truong L, Spagnoli S, Tanguay RL (2018). PLOS ONE 13(3)

AHR2 KO AHR2 KO

5-day developmental tox

AHR2 WT + TCDD

1 ng/mL TCDD

AHR2 KO Mutants Exhibit Reduced AHR2 mRNA Expression Levels and Reduced Expression of Known AHR2 Target Genes

UnexposedExpression

5 dpf

AHR2KO

AHR2KO

AHR2KO

AHR2KO

AHR2KO AHR2

KO AHR2KO

AHR2KO

+TCDD

+TCDD

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Genome-Wide Screens in ToxicologyIdentify mode of action/help define adverse outcome pathway

Define pathways/organelles impactedCell- or tissue-specific mechanisms?

Identify candidate genes for SNP studies—genetic predisposition?Differentiate functionally important genes versus correlation (gene expression)

Allows ranking of function—which are key for survival?Comprehensive assessment of gene products involved in toxicant response

Example of two genome-wide screens to further understand arsenic toxicity

- Arsenics—IARC Group 1 carcinogen- Drinking water exposure- Mechanisms still controversial

(depending on who you ask)

Sobh et al., Toxicological Sciences, 2019

Arsenic Trioxide

Identify genes that modulatesensitivity or resistance

Genome-wide CRISPRIn K562, pre-RBC tumor

Arsenite

Genome-wide CRISPRIn ER-Stress reporter cell line

Identify genes that modulateresponse to arsenic-induced ER stress

Panganiban RA et al., Proc Natl Acad Sci USA. 2019;116(27):13384–93.

How Do You Do Genome-Wide CRISPR Screening?

Pooled sgRNA oligonucleotide

synthesis

Pooled plasmid library

GibsoncloningDesign sgRNA

InfectCells

Lentiviruspackaging

Use webtools todesign sgRNA and

make your own librarye.g., http://crispr.mit.edu/

Or purchase and amplify premade library from Addgene

https://www.addgene.org/crispr/https://www.addgene.org/pooled-library/

Wide variety of libraries availableHumanMouse

Drosophila

Targeting guide Selectable marker

One Vector system

Cas9

Multiple sgRNAper gene

• A different sgRNA in each clone• Multiple copies of each sgRNA• Multiple sgRNA per gene

Clone into lentivirus vector

http://genome-engineering.org/

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Now What?—Make a Mutant KO Library

Pooled LentiCRISPR

sgRNA Library

Package to makeLentiviral

Pool Low MOI Transduction

<1 virus per cell0.3–0.5 MOI

Antibiotic selection

for integrated virus

Library of Mutants

Each cell contains 1 sgRNA+ Cas9

sgRNAtargeted KO

in each cell of one gene

Integrated sgRNA acts as barcode for KO—uniquely identifies cell containing

corresponding KO

Phenotype of Choice

Mutant KO cell library

+ Tox

Control

Reporter

Cell Growth

TOXICANT RESPONSEELEMENT

ENHANCED

REPORTER

DECREASED

Resistant

Sensitive

No Change

Genomic DNA Extraction

QUANTIFICATIONOF KOs in pool

sgRNA Guide PCRfrom each cell

MultiplexSequencing

and Quantification

Mutant KO cell library

+ ToxSort

WT response

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~18,000 genes

Gecko ASingle Vector System

Knockout Library

K562 (human PRE-RED BLOOD

CANCER cell line)

Cell line

Low MOI

PuromycinSelection

Control 7 Cell doublings7 days

Mutant Library

1 mMArsenicTrioxide

ATO refreshedevery 24 hrs

sgRNAPCRNGS

ComputationalAnalysis

Arsenic Trioxide Genome-Wide Growth Screen

2 replicates

Resistant KOs

Sensitive KOs

Each point represents the relative abundance of a sgRNA—3 sgRNA per gene

AsO 3

expo

sed

vs. c

ontro

l

sgRNA counts provide a measure of corresponding mutant cell

abundance in pool

Candidates for 2˚ screen1, 2, or 3 sgRNAFDR Cutoff: 0.1Resistant mutants: 69Sensitive mutants: 37

~IC30-507 days

Primary Screen

Secondary Screen

PathwayAnalysis

Individual Gene Knockout Analysis

Custom validation sgRNA library

6–8 sgRNA /gene~100 arsenic candidates

307 total genes 500 non-targeting sgRNA

2874 total sgRNA

Control 7 Cell doublings

7 days

1 mMArsenicTrioxide

Identical conditionsas 1˚ screen

20% sensitivity candidates

55% of resistance candidates

Confirmed in 2˚ screen

Resistant

Sensitive

Gene Fold Change

sgRNAs q-value (FDR)

KEAP1 5.94 8/8 0.00E+00EEFSEC 2.32 6/8 1.26E-248

TXNDC17 2.19 8/8 3.05E-240PSTK 2.24 7/8 6.98E-222FLCN 1.91 6/8 5.66E-185GFI1B 1.79 7/8 1.53E-170

SEPHS2 1.79 5/8 1.05E-149RRAGC 1.74 7/8 2.65E-146

SEPSECS 1.66 6/8 1.12E-127SLC30A1 1.58 7/8 1.11E-114SECISBP2 1.47 5/8 8.17E-86

Top Resistant KOs

Gene Fold Change sgRNAs q-value

(FDR)ABCC1 0.38 7/8 9.67E-222

NCAPD3 0.65 5/8 2.13E-56UBE2H 0.75 5/8 7.05E-50MTPN 0.66 6/8 4.26E-47NDE1 0.70 7/8 1.05E-43NPRL2 0.75 6/6 1.85E-43CNOT2 0.67 7/7 3.74E-43DEPDC5 0.75 7/7 2.25E-37

DYNC1LI1 0.73 6/7 2.98E-31BPGM 0.83 7/7 1.33E-21

SH3TC1 0.85 8/8 9.22E-18

Top Sensitive KOs

Primary Screen

Secondary Screen

PathwayAnalysis

Individual Gene Knockout Analysis

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KO any gene increases resistance

PathwayAnalysis

Primary Screen

Secondary Screen

SelenocysteineThe 21st amino acid

Individual Gene Knockout Analysis AQP3 MRP1

Zn2+

Zn2+

ATO

ATO/As (III)Se

TrxR1SH SH

SHSe-

SecTRAP

Oxidative Stress

Mitochondrial damage

Nrf2Keap1

DNA Damage& Repair

Protein Damage& Repair

?

Sec

AsGSH

Genome-Wide CRISPR Screen to Identify Arsenite ER Stress-Response Suppressors

As

mCherry fluorescence

even

ts

CHOP Promoter mCherry

As dose

https://doi.org/10.1016/bs.ircmb.2016.10.001

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Panganiban et al., PNAS 2019;116:27:13384–13393

GeckoAB library~18,000 genes6 sgRNA/gene

Three rounds of FACS enrichmentof extreme population

1800 miRNAs

MOI = 0.3

Mutant Library

FACS

Sort forhigh mCHERRY

HyperactiveER stress response

Criteria>2.5 Log FC

>2 sgRNA (out of 6)321 genes37 miRNAs

ASMT—melatonin biosynthesisSLC52A1—riboflavin transporterL3MBTL2—Polycomb transcriptional repressor MB21D2—no known function

CHOP Expression Enrichment Screen

Control

β-actin

IRE1𝝰𝝰

As

- -+ + miR-124 mimic

Test individual KOs mCHERRY reporter

+ L3MBTL2sgRNA

ScrambledsgRNA

Validate3L3MBTL2 MB21D2 SLC52A1

Thapsigargin

L3MBTL2 KO enhances CHOP expression to AS and TG—but not upstream genes

DAPI

L3MBTL2 KO sensitizes cells toarsenic-induced apoptosis

ERSE

DDIT promoter

AP-1CEBP/ATF

AR

AF

BR

BF

CR

CF

DR

DF

0.25

1

4

16

64

256

Rel

ativ

e ex

pres

sion

IgG

L3MBTL2

L3MBTL2 binds to CHOP promoter

Chromatin IP assay

ERstress

CHOP

CHOP

??

ATF4

MGAL3MBTL2

??

MGAL3MBTL2

Apoptosis

ER StressResponseMODEL

Control As0.0

0.5

1.0

1.5

2.0

Rel

ativ

e ex

pres

sion

ScramblemiR-124 mimic

*

*ERN1

miRNA-124 suppresses

the IRE1

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Current CRISPR KO in Toxicology

• Single gene KO studies are already widespread in toxicology •More common in whole organism studies• Likely to be utilized extensively in cell lines/iPSCs• KOs must be subject to same scrutiny as other mutants

•Genome-wide screens emerging in toxicology • Limited to cell lines currently with all caveats• Computational analysis is evolving• Beginning to help define MOA/novel players in toxicology

If I only had legs, I could get out of

this dish

Future Applications of CRISPR in Toxicology • CRISPR variants •CRISPR a/i, multiple Cas enzymes, base editors, epigenetic

modifiers, etc.•Tissue-specific metabolism/tissue-specific effects

• iPSC-derived cells with specific KOs of Phase I–111• CRISPR in metabolically competent cells (e.g., HepaRG) • Conditional CRISPR in whole animals

•CRISPR to infer mode of action/adverse outcome pathway(s)• Genome-wide or targeted libraries • Panel of cell lines/iPSC

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Potential Applications of CRISPR in Toxicant TestingPanel of Individual KOs

in metabolically competent cells

Panel of Differentiated HepaRG

BER

Similar to DT40 Chicken Cell Panelor FANC patient cell lines

DDR MMR DSB

Dose-Response/Viability

ETC

Differential toxicity?Infer insult/MOA

Toxicant

Individual isolated KO for each gene

DNA repair genes

Infer Mode of Action by Functional Profiling

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Toxicant Toxicant

Toxicant

mGene 2

mGene 3

mGene n

Shared key genesSupports hypothesis of similar mode of action

Reference Toxicantswith “known” MOA

Toxicant

FunctionalRequirements

mGene 4

mGene 5

mGene 6

mGene 7

mGene 8

mGene 6

Toxicant

UnknownToxicant

?

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Genome-Wide CRISPR to Infer Adverse Outcome Pathways

mGene 2

mGene 3

mGene n

Identify key set of genes

for toxicant

Adverse Outcome 1

n

3

2

Adverse Outcome 3

Adverse Outcome 2

Map to biological pathways and

processes

Fill in gaps in AOP

Adverse Outcome 1

Adverse Outcome 3

Adverse Outcome 2ToxicantRisk

Assessment

Toxicant

Additional References Start Here: https://www.addgene.org/crispr/

Reviews• Shen H, McHale CM, Smith MT, Zhang L. Functional Genomic Screening Approaches in Mechanistic Toxicology and Potential

Future Applications of CRISPR-Cas9. Mutation Research Reviews in Mutation Research. 2015;764:31–42. • Sobh A, Vulpe C. CRISPR Genomic Screening Informs Gene–Environment Interactions. Current Opinion in Toxicology. 2019;18:46–

53. • Karlgren M, Simoff I, Keiser M, Oswald S, Artursson P. CRISPR-Cas9: A New Addition to the Drug Metabolism and Disposition

Toolbox. Drug Metab Dispos. 2018;46(11):1776–86.

Selected Individual KO Studies• Zagorski JW, Maser TP, Liby KT, Rockwell CE. Nrf2-Dependent and -Independent Effects of tert-Butylhydroquinone, CDDO-Im, and

H2O2 in Human Jurkat T Cells as Determined by CRISPR/Cas9 Gene Editing. J Pharmacol Exp Ther. 2017;361(2):259–67.• Garcia GR, Bugel SM, Truong L, Spagnoli S, Tanguay RL (2018) AHR2 Required for Normal Behavioral Responses and Proper

Development of the Skeletal and Reproductive Systems in Zebrafish. PLOS ONE 13(3): e0193484. • Simoff I, Karlgren M, Backlund M, Lindstrom AC, Gaugaz FZ, Matsson P, Artursson P. Complete Knockout of Endogenous Mdr1

(Abcb1) in MDCK Cells by CRISPR-Cas9. J Pharm Sci. 2016;105(2):1017–21.• Dorr CR, Remmel RP, Muthusamy A, Fisher J, Moriarity BS, Yasuda K, Wu B, Guan W, Schuetz EG, Oetting WS, Jacobson PA, Israni

AK. CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism. Drug Metab Dispos. 2017;45(8):957–65.

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Additional References

• Xia P, Zhang X, Xie Y, Guan M, Villeneuve DL, Yu H. Functional Toxicogenomic Assessment of Triclosan in Human HepG2 Cells Using Genome-Wide CRISPR-Cas9 Screening. Environ Sci Technol. 2016;50(19):10682–92.

• Sundberg CD, Hankinson O. A CRISPR/Cas9 Whole-Genome Screen Identifies Genes Required for Aryl Hydrocarbon Receptor-Dependent Induction of Functional CYP1A1. Toxicol Sci. 2019;170(2):310–9.

• Shortt K, Heruth DP, Zhang N, Wu W, Singh S, Li DY, Zhang LQ, Wyckoff GJ, Qi LS, Friesen CA, Ye SQ. Identification of Novel Regulatory Genes in APAP-Induced Hepatocyte Toxicity by a Genome-Wide CRISPR-Cas9 Screen. Sci Rep. 2019;9(1):1396.

• Tao L, Tian S, Zhang J, Liu Z, Robinson-McCarthy L, Miyashita SI, Breault DT, Gerhard R, Oottamasathien S, Whelan SPJ, Dong M. Sulfated Glycosaminoglycans and Low-Density Lipoprotein Receptor Contribute to Clostridium Difficile Toxin A Entry into Cells. Nat Microbiol. 2019.

• Tao L, Zhang J, Meraner P, Tovaglieri A, Wu X, Gerhard R, Zhang X, Stallcup WB, Miao J, He X, Hurdle JG, Breault DT, Brass AL, Dong M. Frizzled Proteins Are Colonic Epithelial Receptors for C. difficile Toxin B. Nature. 2016;538(7625):350–5.

• Kerins MJ, Liu P, Tian W, Mannheim W, Zhang DD, Ooi A. Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor. Mol Cell Biol. 2019;39(13).

• Panganiban RA, Park HR, Sun M, Shumyatcher M, Himes BE, Lu Q. Genome-Wide CRISPR Screen Identifies Suppressors of Endoplasmic Reticulum Stress-Induced Apoptosis. Proc Natl Acad Sci USA. 2019;116(27):13384–93.

Genome-Wide Screens

Key Concepts/Confusions in Genome-Wide CRISPR Screening

- “In vitro”—using cell lines with all the accompanying issues and caveats- e.g., metabolism, immortalized cells, toxicokinetics

- Any or every gene can be targeted in your library BUT- Only a single gene is inactivated (KO) in each cell- A pool (library) of individual mutant cells each containing a KO of single gene

represents all genes

- The gene on each chromosome are KO’d, but the mutations are different on each chromosome

- Each cell with a KO is TAGGED/FLAGGED with unique DNA barcode (sgRNA) so you can see it in a crowd (pool)

- Generally measuring growth advantage or disadvantage of mutant cells in response to environmental exposure such as toxicant

If I only had legs, I could get out of

this dish

Gene 1KO

Gene 2KO

Gene 3KO

Gene nKO

Xone

1X

two

2X

three

3Xn

n

Xaone

Xbone

1

2

3

n

2

3n

nn n

n

n

n

n

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Presenter 2 | SR01



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