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基于CRISPRCas9技术的基因敲入敲除策略.docx

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基于CRISPRCas9技术的基因敲入敲除策略 Title:CRISPR-Cas9Technology:StrategiesforGeneKnock-inandKnockout Introduction: ThediscoveryofCRISPR-Cas9technologyhasrevolutionizedthefieldofgeneticengineering,providinganefficientandversatiletoolforprecisegenomeediting.Thistechnologyhasopeneduppossibilitiesforawiderangeofapplications,includinggeneknock-inandknock-outstrategies.Inthispaper,wereviewtheprinciplesandadvancementsofCRISPR-Cas9technologyanddiscussvariousstrategiesemployedforgeneknock-inandknock-out. CRISPR-Cas9System: TheCRISPR-Cas9systemisderivedfromtheadaptiveimmunesystemofbacteria,whichusesRNA-guidednucleasestotargetandcleavespecificsequencesofDNA.Cas9,thenucleaseenzyme,isguidedbyasingle-guideRNA(sgRNA)toitstargetsiteinthegenome,whereitintroducesadouble-strandbreak(DSB).Thecellularrepairmechanisms,namelynon-homologousendjoining(NHEJ)andhomology-directedrepair(HDR),canthenbeexploitedtoachievegeneknock-inorknock-out. GeneKnock-outStrategies: Geneknock-outinvolvesdisruptingthefunctionofaspecificgenebyintroducingDSBsnearthetargetgene.TherepairprocessviaNHEJoftenintroducessmallinsertionsordeletions(indels)atthebreaksite,resultinginframeshiftmutationsthatleadtoprematurestopcodonsordysfunctionalproteinproduction.Severalstrategieshavebeendevelopedtoenhancetheefficiencyandspecificityofgeneknock-out,includingtheuseofmultiplesgRNAstargetingdifferentsiteswithinthegeneorutilizingdonorDNAtemplatestofacilitatepreciserepair. GeneKnock-inStrategies: Geneknock-inaimstointroducespecificDNAsequencesintothegenomeatapreciselocation.ThiscanbeachievedbyutilizingtheHDRpathwaytoincorporateexogenousDNAtemplatesintotheDSBsite.Therearetwotypesofknock-instrategies:homologyarm-dependentandhomologyarm-independentknock-in.Homologyarm-dependentknock-inreliesonthepresenceofflankinghomologyarmsinthedonorDNAtemplate,whichfacilitaterecombinationwiththetargetsite.Ontheotherhand,homologyarm-independentknock-in,alsoknownasmicrohomology-mediatedendjoining(MMEJ),utilizesshortstretchesofmicrohomologypresentonbothsidesofth