CRISPR-protocol流程

©2013NatureAmerica,Inc.Allrightsreserved.protocolnatureprotocols|VOL.8NO.11|2013|2281IntroDuctIonTheabilitytoengineerbiologicalsystemsandorganismsholdsenormouspotentialforapplicationsacrossbasicscience,medi-cineandbiotechnology.Programmablesequence-specificendo-nucleasesthatfacilitatepreciseeditingofendogenousgenomiclociarenowenablingsystematicinterrogationofgeneticele-mentsandcausalgeneticvariations1,2inabroadrangeofspecies,includingthosethathavenotpreviouslybeengeneticallytracta-ble3–6.Anumberofgenomeeditingtechnologieshaveemergedinrecentyears,includingzinc-fingernucleases(ZFNs)7–10,transcriptionactivator–likeeffectornucleases(TALENs)10–17andtheRNA-guidedCRISPR-Casnucleasesystem18–25.ThefirsttwotechnologiesuseastrategyoftetheringendonucleasecatalyticdomainstomodularDNA-bindingproteinsforinducingtargetedDNAdouble-strandedbreaks(DSBs)atspe-cificgenomicloci.Bycontrast,Cas9isanucleaseguidedbysmallRNAsthroughWatson-CrickbasepairingwithtargetDNA26–28(Fig.1),representingasystemthatismarkedlyeasiertodesign,highlyspecific,efficientandwell-suitedforhigh-throughputandmultiplexedgeneeditingforavarietyofcelltypesandorganisms.PrecisegenomeeditingusingengineerednucleasesSimilarlytoZFNsandTALENs,Cas9promotesgenomeeditingbystimulatingaDSBatatargetgenomiclocus29,30.UponcleavagebyCas9,thetargetlocustypicallyundergoesoneoftwomajorpathwaysforDNAdamagerepair(Fig.2):theerror-proneNHEJorthehigh-fidelityHDRpathway,bothofwhichcanbeusedtoachieveadesirededitingoutcome.Intheabsenceofarepairtem-plate,DSBsarere-ligatedthroughtheNHEJprocess,whichleavesscarsintheformofinsertion/deletion(indel)mutations.NHEJcanbeharnessedtomediategeneknockouts,asindelsoccurringwithinacodingexoncanleadtoframeshiftmutationsandprema-turestopcodons31.MultipleDSBscanadditionallybeexploitedtomediatelargerdeletionsinthegenome22,32.HDRisanalternativemajorDNArepairpathway.AlthoughHDRtypicallyoccursatlowerandsubstantiallymorevariablefrequenciesthanNHEJ,itcanbeleveragedtogenerateprecise,definedmodificationsatatargetlocusinthepresenceofanexo-genouslyintroducedrepairtemplate.Therepairtemplatecaneitherbeintheformofconventionaldouble-strandedDNAtargetingconstructswithhomologyarmsflankingtheinsertionsequence,orsingle-strandedDNAoligonucleotides(ssODNs).Thelatterprovidesaneffectiveandsimplemethodformakingsmalleditsinthegenome,suchastheintroductionofsingle-nucleotidemutationsforprobingcausalgeneticvariations32.UnlikeNHEJ,HDRisgenerallyactiveonlyindividingcells,anditsefficiencycanvarywidelydependingonthecelltypeandstate,aswellasthegenomiclocusandrepairtemplate33.Cas9:anRNA-guidednucleaseforgenomeeditingCRISPR-CasisamicrobialadaptiveimmunesystemthatusesRNA-guidednucleasestocleaveforeigngeneticelements18–21,26.Threetypes(I–III)ofCRISPRsystemshavebeenidentifiedacrossawiderangeofbacterialandarchaealhosts,whereineachsystemcomprisesaclusterofCRISPR-associated(Cas)genes,noncodingRNAsandadistinctivearrayofrepetitiveelements(directrepeats).Theserepeatsareinterspacedbyshortvariablesequences20derivedfromexogenousDNAtargetsknownasprotospacers,andtogethertheyconstitutetheCRISPRRNA(crRNA)array.WithintheDNAtarget,eachprotospacerisalwaysassociatedwithaprotospaceradjacentmotif(PAM),whichcanvarydependingonthespecificCRISPRsystem34–36.TheTypeIICRISPRsystemisoneofthebestcharacterized26–28,37,38,consistingofthenucleaseCas9,thecrRNAarraythatencodestheguideRNAsandarequiredauxiliarytrans-activatingcrRNA(tracrRNA)thatfacilitatestheprocessingofthecrRNAarrayintodiscreteunits26,28.EachcrRNAunitthencontainsa20-ntguidesequenceandapartialdirectrepeat,wheretheformerdirectsCas9toa20-bpDNAtargetviaWatson-Crickbasepair-ing(Fig.1).IntheCRISPR-CassystemderivedfromStreptococcuspyogenes(whichisthesystemusedinthisprotocol),thetargetDNAmustimmediatelyprecedea5′-NGGPAM27,whereasGenomeengineeringusingtheCRISPR-Cas9systemFAnnRan1–5,8,PatrickDHsu1–5,8,JasonWright1,VineetaAgarwala1,6,7,DavidAScott1–4&FengZhang1–41BroadInstituteofMassachusettsInstituteofTechnology(MIT)andHarvard,Cambridge,Massachusetts,USA.2McGovernInstituteforBrainResearch,Cambridge,Massachusetts,USA.3DepartmentofBrainandCognitiveSciences,MIT,Cambridge,Massachusetts,USA.4DepartmentofBiologicalEngineering,MIT,Cambridge,Massachusetts,USA.5DepartmentofMolecularandCellularBiology,HarvardUniversity,Cambridge,Massachusetts,USA.6PrograminBiophysics,HarvardUniversity,MIT,Cambridge,Massachusetts,USA.7Harvard-MITDivisionofHealthSciencesandTechnology,MIT,Cambridge,Massachusetts,USA.8Theseauthorscontributedequallytothiswork.CorrespondenceshouldbeaddressedtoF.Z.([e-mail]).Publishedonline24October2013;doi:10.1038/nprot.2013.143targetednucleasesarepowerfultoolsformediatinggenomealterationwithhighprecision.therna-guidedcas9nucleasefromthemicrobialclusteredregularlyinterspacedshortpalindromicrepeats(crIspr)adaptiveimmunesystemcanbeusedtofacilitateefficientgenomeengineeringineukaryoticcellsbysimplyspecifyinga20-nttargetingsequencewithinitsguiderna.Herewedescribeasetoftoolsforcas9-mediatedgenomeeditingvianonhomologousendjoining(nHeJ)orhomology-directedrep