Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same...
Transcript of Genome edi3ng with the CRISPR-Cas9 system · CRISPR-Cas9 • Cas9 nuclease remains the same...
CRISPR-Cas9GenomeEdi3ngBootcampAHACouncilonFunc3onalGenomicsandTransla3onalBiology
Narratedvideolink:hFps://youtu.be/h18HmFtybnQ
Genomeedi3ngwiththeCRISPR-Cas9system
KiranMusunuru,MD,PhD,MPH,FAHA
FINANCIALDISCLOSURES:NoneUNLABELED/UNAPPROVEDUSESDISCLOSURES:None
Genomeedi3ng
•Facilitatesknockoutorknock-inofmuta3onsbyintroducingadouble-strandbreakatadesiredsiteingenome
•Drama3callyincreasestheefficiencyofmutagenesis
•Canbeusedinvitroandinvivo
Double-strandbreaks
•Thecellhastwomethodstorepairdouble-strandbreaks(DSBs)
•Non-homologousendjoining(NHEJ)–bringstwofreeendstogetherandrejoinsthem,error-prone
•Homology-directedrepair(HDR)–usessisterchroma3d/chromosomeasatemplatetoreplacetheareaofthebreakviahomologousrecombina3on
Double-strandbreaks
•CanfoolthecellintousinganexogenouslyintroducedDNAvectorasarepairtemplate
•Canevenuseasingle-strandDNAoligonucleo3de(ssDNAoligo)asarepairtemplate
•IfavectororssDNAoligoharborsamuta3on,canexploitHDRtostablyintroducethemuta3onintothegenome
Double-strandbreaks
Gupta and Musunuru. J Clin Invest 2014; 124:4154-61
Genome-edi3ngtools
•Zincfingernucleases(ZFNs)
•Meganucleases
•TALeffectornucleases(TALENs)
•Clusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)–CRISPR-associated9(CRISPR-Cas9)
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)
CRISPR-Cas9
•Cas9nucleaseremainsthesameregardlessoftargetDNA
•Changing20-21nucleo3desintheguideRNAaltersthesequencespecificityoftheCRISPR-Cas9complex
•CanmakeanewguideRNAinthelaboratoryinoneday
•Canmakealargelibrary(genome-wide)atone3me
•MixingCas9withmorethanoneguideRNAallowsformul3plexing–targe3ngmul3plesitesatonce
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
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)
Genera3ngknockoutmicewithCRISPR-Cas9
mutagenesis
zygoteinjec3on blastocyst
embryotransfer
AAAA
Cas9mRNA,guideRNA(gRNA)
PCRscreening
knockoutmice
3weekstomakeknockoutmiceupto100%efficiency
DiseasemodelinginstemcellswithCRISPR-Cas9
mutanthPSCor
correctediPSC
genomeedi3ng
wild-typehPSCor
iPSCwithmuta3on
differen3a3on
phenotypiccomparisons
iPSC=inducedpluripotentstemcells
hPSC=humanpluripotentstemcells(iPSCorhumanembryonicstemcells)
On-targetvs.off-targeteffects
•CRISPR-Cas9canbeveryefficient–asmuchas100%mutagenesisinsomeapplica3ons
•Alongwithon-targetmutagenesis,therecanbeoff-targetmutagenesiselsewhereinthegenome
•Off-targeteffectsthoughttobemostlikelytohappenatsiteswithsequencesimilaritytoon-targetsite
•StreptococcuspyogenesCas9
- the“standard”Cas9usedforvirtuallyallresearchapplica3onstodate
- well-characterizedon-target,off-targeteffects
•StaphylococcusaureusCas9
- smallerthanS.pyogenesCas9,canbepackagedinAAVforinvivodelivery
- similaron-targetefficiency,possiblylessoff-targeteffects(Ranetal.,Nature2015)
CRISPR-Cas9systems
Introduc3onofCas9andguideRNAintocells
guideRNAU6promoter G(N)20
LigatetwoshortoligosintoplasmidencodingguideRNA=pGuide
CAGpromoter 2A GFPCas9(fromS.pyogenes)
FixedplasmidencodingCas9andgreenfluorescentprotein(GFP)=pCas9_GFP
Streptococcuspyogenes
PlasmidsavailablefromAddgene;otherS.pyogenesCRISPR-Cas9plasmidsareavailable
Introduc3onofCas9andguideRNAintocells
guideRNAU6promoter G(N)21
LigatetwoshortoligosintoplasmidencodingguideRNA=pSaGuide
CAGpromoter 2A GFPCas9(fromS.aureus)
FixedplasmidencodingCas9andgreenfluorescentprotein(GFP)=pSaCas9_GFP
Staphylococcusaureus
PlasmidsavailablefromAddgene;otherS.aureusCRISPR-Cas9plasmidsareavailable
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
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
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
•Iftryingtoknockoutagene,pickatargetsiteinthefirstcodingexon(or,ifthegeneappearstohavemul3plealterna3vetranscriptsinUCSCGenomeBrowser,picktheearliestcodingexonthatissharedbyalltranscripts)
•Iftryingtoknockinavariant,thesiteselec3onwillbeconstrained–shouldbewithin10-15bpofthedesiredvariant,andideallyless
Tipsforiden3fyingtargetsite
•UsecDNAsequence(con3nuouscodingsequence)todeterminethetargetsiteandtheconsequenceofamuta3on–candownloadfromGenBank
•Usegenomesequencetoiden3fyop3malprotospacer/PAMsites–candownloadfromUCSCGenomeBrowser
•IfcDNAsequenceisusedtofindprotospacer/PAMsites,willnotaccountforintronicsequences–maynotsuccessfullytargetthegenome
Tipsforiden3fyingtargetsite
•Familialcombinedhypolipidemia
•Loss-of-func3onmuta3onsintheANGPTL3gene
•S17Xmuta3on(Ser17Ter)isthemostcommonlyreported
•Task:designS.pyogenesguideRNAstotargetthesiteofthemuta3on,bothforknockoutoftheANGPTL3geneaswellastoknockinthespecificmuta3on
Example
ANGPTL3codingsequence(fromgenomesequence)
ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...
Example
S17Xmuta3on=TCC!TGA
No“NGG”sequences(PAMs)nearthemuta3onsite
Mul3ple“CCN”sequences(reversePAMs)
ANGPTL3codingsequence(fromgenomesequence)
ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...
Example
PAM#1:protospacer=ATTCTGGAGGAAATAACTAG
Double-strandbreakoccurs10bpawayfrommuta3onsite,protospaceroverlapsmuta3onsite(reducingre-cleavageofsite,oncemuta3onisintroduced)
•Overlapofprotospacerand/orPAMwiththemuta3onsiteresultsinamismatch(es)aeerknock-in
•Oneortwoprotospacermismatchesshouldreducebutmaynoteliminatere-cleavage
•ProtospacermismatchesclosetoPAMwillgenerallyreducere-cleavagemorethanthoseawayfromPAM
•Disrup3onofPAM(nolongerNGG)shouldeliminatere-cleavage
Reducingre-cleavagebyCRISPR-Cas9
ANGPTL3codingsequence(fromgenomesequence)
ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...
Example
PAM#2:protospacer=ATTGTCTTGATCAATTCTGG
Double-strandbreakoccurs1bpawayfrommuta3onsite,protospaceroverlapsmuta3onsite(reducingre-cleavageofsite,oncemuta3onisintroduced)
ANGPTL3codingsequence(fromgenomesequence)
ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...
Example
PAM#3:protospacer=TGAATTGTCTTGATCAATTC
Double-strandbreakoccurs4bpawayfrommuta3onsite,PAMoverlapsmuta3onsite(reducingre-cleavageofsite,oncemuta3onisintroduced)
ANGPTL3codingsequence(fromgenomesequence)
ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...
Example
PAM#2:protospacer=ATTGTCTTGATCAATTCTGG
Bestchoiceonpaper,butideallyallthreecandidatesshouldbetestedforDSBac3vityinahumancellline(e.g.,HEK293cells)toiden3fythebestguideRNA
PAM#2:protospacer=ATTGTCTTGATCAATTCTGG
Todesignoligonucleo3destoinsertintopGuide:
First,add“G”tothebeginningoftheprotospacertoallowtranscrip3onbyU6promoter:
GATTGTCTTGATCAATTCTGG
Thisisthesequencethatneedstobeincorporatedatthe5’endoftheguideRNA
Example
GATTGTCTTGATCAATTCTGG
Todesignoligonucleo3destoinsertintopGuide:
Second,usethefollowingtemplates:
5’-CACC-GNNNNNNNNNNNNNNNNNNNN-3’
3’-CNNNNNNNNNNNNNNNNNNNN-CAAA-5’
ThesetwooligoscanbeannealedtogethertoformasmallinsertthatcanbeligatedintopGuide
Example
GATTGTCTTGATCAATTCTGG
Templates:
5’-CACC-GNNNNNNNNNNNNNNNNNNNN-3’
3’-CNNNNNNNNNNNNNNNNNNNN-CAAA-5’
Oligonucleo3des(canbuythese):
5’-CACCGATTGTCTTGATCAATTCTGG-3’
5’-AAACCCAGAATTGATCAAGACAATC-3’
Example
•Uponcrea3onofthepGuidevectorwiththeguideRNAtarge3ngANGPTL3S17Xsite,canbeusedfor:
- knockout:sincethesiteissoclosetothebeginningofthegene,anyindelwilltruncatetheprotein
- knock-inofmuta3on:ifusedwithasingle-strandDNAoligonucleo3deasarepairtemplate,HDRwillallowforknock-inatalowfrequency;however,NHEJwills3llbeac3veandresultinindels/knockoutsatsomefrequency
Example
•Todesignsingle-strandDNAoligonucleo3deforknock-in,startwiththemutantnucleo3de(s)andthenaddflankinggenomesequencesoneithersideofthesitetoserveashomologyarms
•Shouldusehomologyarms≥40nucleo3desinlength
•CanusessDNAoligosaslongas200nucleo3des
Example
ANGPTL3codingsequence(fromgenomesequence)
ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...
Example
S17Xmuta3on=TCC!TGA
ssDNAoligo(canbuythis)=5’-ATTAAGCTCCTTCTT TTTATTGTTCCTCTAGTTATTTCCTGAAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTC-3’
•ThelaststepistodesignPCRprimerstoamplifytheregionsurroundingthetargetsite(severalhundredbasepairlengthissufficient,aslongasthetargetsiteisinthecenteroftheamplicon)
•CELIendonucleaseorT7endonucleaseI(T7EI)assay(detectsDNAmismatcheswithinaPCRproduct)todeterminetheefficacyofmutagenesis
•DNAsequencingtoiden3fyspecificmuta3ons
Assessingefficacy