Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same...

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CRISPR-Cas9 Genome Edi3ng Bootcamp AHA Council on Func3onal Genomics and Transla3onal Biology Narrated video link: hFps://youtu.be/h18HmFtybnQ Genome edi3ng with the CRISPR-Cas9 system Kiran Musunuru, MD, PhD, MPH, FAHA FINANCIAL DISCLOSURES: None UNLABELED/UNAPPROVED USES DISCLOSURES: None

Transcript of Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same...

Page 1: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

CRISPR-Cas9GenomeEdi3ngBootcampAHACouncilonFunc3onalGenomicsandTransla3onalBiology

Narratedvideolink:hFps://youtu.be/h18HmFtybnQ

Genomeedi3ngwiththeCRISPR-Cas9system

KiranMusunuru,MD,PhD,MPH,FAHA

FINANCIALDISCLOSURES:NoneUNLABELED/UNAPPROVEDUSESDISCLOSURES:None

Page 2: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

Genomeedi3ng

•Facilitatesknockoutorknock-inofmuta3onsbyintroducingadouble-strandbreakatadesiredsiteingenome

•Drama3callyincreasestheefficiencyofmutagenesis

•Canbeusedinvitroandinvivo

Page 3: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

Double-strandbreaks

•Thecellhastwomethodstorepairdouble-strandbreaks(DSBs)

•Non-homologousendjoining(NHEJ)–bringstwofreeendstogetherandrejoinsthem,error-prone

•Homology-directedrepair(HDR)–usessisterchroma3d/chromosomeasatemplatetoreplacetheareaofthebreakviahomologousrecombina3on

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Double-strandbreaks

•CanfoolthecellintousinganexogenouslyintroducedDNAvectorasarepairtemplate

•Canevenuseasingle-strandDNAoligonucleo3de(ssDNAoligo)asarepairtemplate

•IfavectororssDNAoligoharborsamuta3on,canexploitHDRtostablyintroducethemuta3onintothegenome

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Double-strandbreaks

Gupta and Musunuru. J Clin Invest 2014; 124:4154-61

Page 6: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

Genome-edi3ngtools

•Zincfingernucleases(ZFNs)

•Meganucleases

•TALeffectornucleases(TALENs)

•Clusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)–CRISPR-associated9(CRISPR-Cas9)

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TheCRISPR-Cas9systemforgenomeedi3ng

Mali et al. Science 2013; 339:823-6

Cong et al. Science 2013; 339:819-23

Jinek et al. eLife 2013; 2:e00471

(guide RNA)

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CRISPR-Cas9

•Cas9nucleaseremainsthesameregardlessoftargetDNA

•Changing20-21nucleo3desintheguideRNAaltersthesequencespecificityoftheCRISPR-Cas9complex

•CanmakeanewguideRNAinthelaboratoryinoneday

•Canmakealargelibrary(genome-wide)atone3me

•MixingCas9withmorethanoneguideRNAallowsformul3plexing–targe3ngmul3plesitesatonce

Page 9: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

KnockingoutgeneswithCRISPR-Cas9

CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAACGACTGATGCACCAGAT GCCTCACATGGTGGGGATCGTTTG \ / CACAGTGGTGCTAAACGTGC

20-bpprotospacer

GTGTCACCACGTAATGCACGguideRNAS.pyogenes

Cas9protein G

??????GAGTGTACCACCCCTAGCAAACCTGACTACGTGGTCTAGTGT???????????? ??????CTCACATGGTGGGGATCGTTTGGACTGATGCACCAGATCACA????????????

Genera3onofadouble-strandbreakatgenomicsite

CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAACCTGACTACGTGGTCTAGTGTCACCACGTAA---ACGCGGAGTGTACCACCCCTAGCAAACCTGACTACGTGGTCTAGTGTCACCACGT-----ACGCGGAGTGTACCACCCCTAGCAAACCTGACTACGTGGTCTAGTGTCACCACG-------CGCGGAGTGTACCACCCCTAGCAAAC

wild-typevs.indels/frameshies

Non-homologousendjoining

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KnockinginvariantswithCRISPR-Cas9

CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAACGACTGATGCACCAGAT GCCTCACATGGTGGGGATCGTTTG \ / CACAGTGGTGCTAAACGTGC

20-bpprotospacer

GTGTCACCACGTAATGCACGguideRNAS.pyogenes

Cas9protein G

??????GAGTGTACCACCCCTAGCAAACCTGACTACGTGGTCTAGTGT???????????? ??????CTCACATGGTGGGGATCGTTTGGACTGATGCACCAGATCACA????????????

Genera3onofadouble-strandbreakatgenomicsite

CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAACCTGACTACGTGGTCTAGTGTCACCACGTAATTCACGCGGAGTGTACCACCCCTAGCAAAC

wild-typevs.site-specificmutagenesis

Homology-directedrepairusingDNAtemplate(double-strandvector,ssDNAoligo)

Page 11: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

Genera3ngknockoutmicewithCRISPR-Cas9

mutagenesis

zygoteinjec3on blastocyst

embryotransfer

AAAA

Cas9mRNA,guideRNA(gRNA)

PCRscreening

knockoutmice

3weekstomakeknockoutmiceupto100%efficiency

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DiseasemodelinginstemcellswithCRISPR-Cas9

mutanthPSCor

correctediPSC

genomeedi3ng

wild-typehPSCor

iPSCwithmuta3on

differen3a3on

phenotypiccomparisons

iPSC=inducedpluripotentstemcells

hPSC=humanpluripotentstemcells(iPSCorhumanembryonicstemcells)

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On-targetvs.off-targeteffects

•CRISPR-Cas9canbeveryefficient–asmuchas100%mutagenesisinsomeapplica3ons

•Alongwithon-targetmutagenesis,therecanbeoff-targetmutagenesiselsewhereinthegenome

•Off-targeteffectsthoughttobemostlikelytohappenatsiteswithsequencesimilaritytoon-targetsite

Page 14: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

•StreptococcuspyogenesCas9

- the“standard”Cas9usedforvirtuallyallresearchapplica3onstodate

- well-characterizedon-target,off-targeteffects

•StaphylococcusaureusCas9

- smallerthanS.pyogenesCas9,canbepackagedinAAVforinvivodelivery

- similaron-targetefficiency,possiblylessoff-targeteffects(Ranetal.,Nature2015)

CRISPR-Cas9systems

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Introduc3onofCas9andguideRNAintocells

guideRNAU6promoter G(N)20

LigatetwoshortoligosintoplasmidencodingguideRNA=pGuide

CAGpromoter 2A GFPCas9(fromS.pyogenes)

FixedplasmidencodingCas9andgreenfluorescentprotein(GFP)=pCas9_GFP

Streptococcuspyogenes

PlasmidsavailablefromAddgene;otherS.pyogenesCRISPR-Cas9plasmidsareavailable

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Introduc3onofCas9andguideRNAintocells

guideRNAU6promoter G(N)21

LigatetwoshortoligosintoplasmidencodingguideRNA=pSaGuide

CAGpromoter 2A GFPCas9(fromS.aureus)

FixedplasmidencodingCas9andgreenfluorescentprotein(GFP)=pSaCas9_GFP

Staphylococcusaureus

PlasmidsavailablefromAddgene;otherS.aureusCRISPR-Cas9plasmidsareavailable

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Targe3ngasiteinthegenome

RulesforS.pyogenesCRISPRguideRNAdesign:

1.  Theprotospaceris20basepairsinlength2.  Theprotospacermustbeposi3onedjustbeforea3-bpelementmatchingthe

sequenceNGG(N=anynucleo3de)–calledtheprotospacer-adjacentmo3f(PAM)

3.  The5’por3onoftheguideRNAmatchestheprotospacersequence,sothatitcanbindtothecomplementarystrandofDNA

4.  TobeexpressedefficientlyfromtheU6promoter,theguideRNAsequencemustbeginwitha“G”

5.  Thus,theguideRNAshouldstartwith“G”followedbythe20-bpprotospacer=G(N)20

CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAACGACTGATGCACCAGAT GCCTCACATGGTGGGGATCGTTTG \ / CACAGTGGTGCTAAACGTGC

20-bpprotospacer

GTGTCACCACGTAATGCACGguideRNAS.pyogenes

Cas9protein G

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Targe3ngasiteinthegenome

Pickingatargetsite:

1.  Thedouble-strandbreakoccurs3basepairsupstreamofthePAM2.  Muta3onstendtooccurrightatthesiteofthedouble-strandbreak3.  Cas9–guideRNAcomplexescanbindoneitherDNAstrand,socheckboththe

forwardandreversestrandsforpossibleprotospacers/PAMs

4.  TrytopickaguideRNAsequencethatwillposi3onthedouble-strandbreakascloseaspossibletothesiteofthedesiredmuta3on(whetheranindeloraknock-invariant)

5.  Oeentherewillbeseveralreasonablechoices6.  Ifpossible,avoidGC-richprotospacers(increasedchanceofoff-targeteffects)

break

CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAACGACTGATGCACCAGAT GCCTCACATGGTGGGGATCGTTTG \ / CACAGTGGTGCTAAACGTGC

20-bpprotospacer

GTGTCACCACGTAATGCACGguideRNAS.pyogenes

Cas9protein G

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Targe3ngasiteinthegenome

RulesforS.aureusCRISPRguideRNAdesign:

1.  SimilartorulesforS.pyogenesCRISPRdesign,but…2.  Theprotospaceris21basepairsinlength3.  Theprotospacershouldbeposi3onedjustbeforea5-bpelementmatchingthe

PAMsequenceNNGRR(N=anynucleo3de,R=GorA),ideallyNNGRRT

4.  The5’por3onoftheguideRNAmatchestheprotospacersequence,sothatitcanbindtothecomplementarystrandofDNA

5.  TobeexpressedefficientlyfromtheU6promoter,theguideRNAsequencemustbeginwitha“G”

6.  Thus,theguideRNAshouldstartwith“G”followedbythe21-bpprotospacer=G(N)21

CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAACGACTGATGCACCAGA GCCTCACATGGTGGGGATCGTTTG \ / TCACAGTGGTGCTAAACGTGC

21-bpprotospacerguideRNAS.aureus

Cas9protein AGTGTCACCACGTAATGCACGG

break

Page 20: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

•Iftryingtoknockoutagene,pickatargetsiteinthefirstcodingexon(or,ifthegeneappearstohavemul3plealterna3vetranscriptsinUCSCGenomeBrowser,picktheearliestcodingexonthatissharedbyalltranscripts)

•Iftryingtoknockinavariant,thesiteselec3onwillbeconstrained–shouldbewithin10-15bpofthedesiredvariant,andideallyless

Tipsforiden3fyingtargetsite

Page 21: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

•UsecDNAsequence(con3nuouscodingsequence)todeterminethetargetsiteandtheconsequenceofamuta3on–candownloadfromGenBank

•Usegenomesequencetoiden3fyop3malprotospacer/PAMsites–candownloadfromUCSCGenomeBrowser

•IfcDNAsequenceisusedtofindprotospacer/PAMsites,willnotaccountforintronicsequences–maynotsuccessfullytargetthegenome

Tipsforiden3fyingtargetsite

Page 22: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

•Familialcombinedhypolipidemia

•Loss-of-func3onmuta3onsintheANGPTL3gene

•S17Xmuta3on(Ser17Ter)isthemostcommonlyreported

•Task:designS.pyogenesguideRNAstotargetthesiteofthemuta3on,bothforknockoutoftheANGPTL3geneaswellastoknockinthespecificmuta3on

Example

Page 23: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

ANGPTL3codingsequence(fromgenomesequence)

ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...

Example

S17Xmuta3on=TCC!TGA

No“NGG”sequences(PAMs)nearthemuta3onsite

Mul3ple“CCN”sequences(reversePAMs)

Page 24: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

ANGPTL3codingsequence(fromgenomesequence)

ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...

Example

PAM#1:protospacer=ATTCTGGAGGAAATAACTAG

Double-strandbreakoccurs10bpawayfrommuta3onsite,protospaceroverlapsmuta3onsite(reducingre-cleavageofsite,oncemuta3onisintroduced)

Page 25: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

•Overlapofprotospacerand/orPAMwiththemuta3onsiteresultsinamismatch(es)aeerknock-in

•Oneortwoprotospacermismatchesshouldreducebutmaynoteliminatere-cleavage

•ProtospacermismatchesclosetoPAMwillgenerallyreducere-cleavagemorethanthoseawayfromPAM

•Disrup3onofPAM(nolongerNGG)shouldeliminatere-cleavage

Reducingre-cleavagebyCRISPR-Cas9

Page 26: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

ANGPTL3codingsequence(fromgenomesequence)

ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...

Example

PAM#2:protospacer=ATTGTCTTGATCAATTCTGG

Double-strandbreakoccurs1bpawayfrommuta3onsite,protospaceroverlapsmuta3onsite(reducingre-cleavageofsite,oncemuta3onisintroduced)

Page 27: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

ANGPTL3codingsequence(fromgenomesequence)

ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...

Example

PAM#3:protospacer=TGAATTGTCTTGATCAATTC

Double-strandbreakoccurs4bpawayfrommuta3onsite,PAMoverlapsmuta3onsite(reducingre-cleavageofsite,oncemuta3onisintroduced)

Page 28: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

ANGPTL3codingsequence(fromgenomesequence)

ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...

Example

PAM#2:protospacer=ATTGTCTTGATCAATTCTGG

Bestchoiceonpaper,butideallyallthreecandidatesshouldbetestedforDSBac3vityinahumancellline(e.g.,HEK293cells)toiden3fythebestguideRNA

Page 29: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

PAM#2:protospacer=ATTGTCTTGATCAATTCTGG

Todesignoligonucleo3destoinsertintopGuide:

First,add“G”tothebeginningoftheprotospacertoallowtranscrip3onbyU6promoter:

GATTGTCTTGATCAATTCTGG

Thisisthesequencethatneedstobeincorporatedatthe5’endoftheguideRNA

Example

Page 30: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

GATTGTCTTGATCAATTCTGG

Todesignoligonucleo3destoinsertintopGuide:

Second,usethefollowingtemplates:

5’-CACC-GNNNNNNNNNNNNNNNNNNNN-3’

3’-CNNNNNNNNNNNNNNNNNNNN-CAAA-5’

ThesetwooligoscanbeannealedtogethertoformasmallinsertthatcanbeligatedintopGuide

Example

Page 31: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

GATTGTCTTGATCAATTCTGG

Templates:

5’-CACC-GNNNNNNNNNNNNNNNNNNNN-3’

3’-CNNNNNNNNNNNNNNNNNNNN-CAAA-5’

Oligonucleo3des(canbuythese):

5’-CACCGATTGTCTTGATCAATTCTGG-3’

5’-AAACCCAGAATTGATCAAGACAATC-3’

Example

Page 32: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

•Uponcrea3onofthepGuidevectorwiththeguideRNAtarge3ngANGPTL3S17Xsite,canbeusedfor:

-  knockout:sincethesiteissoclosetothebeginningofthegene,anyindelwilltruncatetheprotein

-  knock-inofmuta3on:ifusedwithasingle-strandDNAoligonucleo3deasarepairtemplate,HDRwillallowforknock-inatalowfrequency;however,NHEJwills3llbeac3veandresultinindels/knockoutsatsomefrequency

Example

Page 33: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

•Todesignsingle-strandDNAoligonucleo3deforknock-in,startwiththemutantnucleo3de(s)andthenaddflankinggenomesequencesoneithersideofthesitetoserveashomologyarms

•Shouldusehomologyarms≥40nucleo3desinlength

•CanusessDNAoligosaslongas200nucleo3des

Example

Page 34: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

ANGPTL3codingsequence(fromgenomesequence)

ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...

Example

S17Xmuta3on=TCC!TGA

ssDNAoligo(canbuythis)=5’-ATTAAGCTCCTTCTT TTTATTGTTCCTCTAGTTATTTCCTGAAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTC-3’

Page 35: Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same regardless of target DNA • Changing 20-21 nucleo3des in the guide RNA alters the sequence

•ThelaststepistodesignPCRprimerstoamplifytheregionsurroundingthetargetsite(severalhundredbasepairlengthissufficient,aslongasthetargetsiteisinthecenteroftheamplicon)

•CELIendonucleaseorT7endonucleaseI(T7EI)assay(detectsDNAmismatcheswithinaPCRproduct)todeterminetheefficacyofmutagenesis

•DNAsequencingtoiden3fyspecificmuta3ons

Assessingefficacy