Lecture CRISPR Cas9技術(shù)講解課件

CRISPR-Cas9TechniqueIntroductionofCRISPR-Cas9techniqueJenniferDoudna,right,saysthatsheandherlabmanager,KaihongZhou,are“twopeasinapod.”Science.2013Feb15;339(6121):819-23.張鋒ZhangFeng1983年生于中國，幼年時(shí)隨家庭移民到美國艾奧瓦州，在此長大。美國神經(jīng)生物學(xué)家，現(xiàn)任麻省理工學(xué)院助理教授。2004年，在哈佛大學(xué)取得化學(xué)及物理學(xué)士。2009年，在斯坦福大學(xué)取得化學(xué)及生物工程博士。2013年，他的實(shí)驗(yàn)室發(fā)展出CRISPR/Cas系統(tǒng)，可以用來編輯DNA，敲除指定的基因。2014年，被《自然》雜志評選為2013年年度十大科學(xué)人物之一。CRISPR——ClusteredRegularlyInterspacedShortPalindromicRepeatsCas——CRISPR-associatedProtospacer(DNA)PAM——ProtospacerAdjacentMotiff(DNA)spacer（RNA）CrRNA——CRISPRrelatedRNAtracrRNA——trans-activatingRNAsgRNA——singleguidedRNACas9Science.2012,17;337(6096):816-21.CollaborationwithOsamuNurekiandHiroshiNishimasuatUniversityofTokyorevealscrystalstructureofCas9incomplexwithguideRNAandtargetDNA.cell.Volume156,Issue5,p935–949,27February2014.Cas9HNHdomaincleavesthecomplementaryDNAstrandCas9RuvC-likedomaincleavesthenoncomplementaryDNAstrand23ntSpCas9SpCas9PAMNNNThenoncomplementaryDNAstrandiscleavedatoneormoresiteswithinthreetoeightbasepairsupstreamofthePAM.Thenoncomplementarystrandisfirstcleavedendonucleolyticallyandsubsequentlytrimmedbya3′-5′exonucleaseactivity(?).5′5′3′3′efficientbutlessprecisesinglecas9cutTheDNAstrandthatiscomplementarytothetarget-bindingsequenceinthecrRNA(thecomplementarystrand)iscleavedatasitethreebasepairsupstreamofthePAM).PAMNNN……moreprecisebutlessefficientdoublecas9s（D10A）cutsCrRNAandtracrRNAsgRNAOff-TargetPositiondependent:the8-14bponthe3′endoftheguidesequenceislesstolerantofmismatches

thanthe5′bases;Quantitydependent:ingeneral,morethanthreemismatchesarenottolerated;Guidesequencedependent:someguidesequencesarelesstolerantofmismatchesthanothers;Concentrationdependent:off-targetcleavageishighlysensitivetothetransfectedamounts,aswellasrelativeratiosofCas9andsgRNA.Minimizationofoff-targetsthroughcrRNAsequenceoptimization

TobeexpatiatedbyYuanPotential‘off-target’genomicsequencesshouldbeidentifiedbyconsideringthefollowingfourconstraints：Firstandforemost,theyshouldnotbefollowedbyaPAM.Second,theirglobalsequencesimilaritytothetargetsequenceshouldbeminimized,andguidesequenceswithgenomicoff-targetlocithathavefewerthanthreemismatchesshouldbeavoided.(?)Third,atleasttwomismatchesshouldliewithinthePAM-proximalregionoftheoff-targetsite.(?)Fourth,amaximalnumberofmismatchesshouldbeconsecutiveorspacedlessthanfourbasesapart.Finally,theamountofSpCas9andsgRNAcanbetitratedtooptimizeon-tooff-targetcleavageratio.FORtheCONSTRUCTIONOFVECTOR

Introductionofgenestructureandtranscription+1:startingpoint-10-35promoterRBSORFmRNAtranscriptionproteinρ-(非)依賴的轉(zhuǎn)錄終止結(jié)構(gòu)terminatortranslationoperorator+1-10-35promoternon-codingRNAtranscriptionρ-independenttranscriptionalterminatorpromotersNucleicAcidsResearch,1997,25(6):1203–1210terminatorsThenucleotidesequenceofP.aeruginosaPAO1rsmB.ResearchinMicrobiology156(2005)7–16ThenucleotidesequenceofP.aeruginosaPAO1rsmZ.JOURNALOFBACTERIOLOGY,2004,2936–2945PreparationofcrRNAandtracrRANSeparatedstyleCombinatorlystyle: ChimericguideRNA

singleguideRNA，sgRNA

synthesiziedRNAScience.2012Aug17;337(6096):816-21Science.2013Feb15;339(6121):819-23.SeparatedstyleNatBiotechnol.2013Mar;31(3):233-9.BbsI5'...GGTCTCNNNNNNN...3'3'...CCAGAGNNNNNNN...5'one-sgRNAbackboneBsaI5'...GGTCTCN...3'3'...CCAGAGNNNNN...5'BsaI5'...GGTCTCN...3'3'...CCAGAGNNNNN...5'……TGATAGAGATACTGAGCACgagaccaaaGGTCTCg

ttttagagctaGAAAtagcaagtt……

…………CTCGTGgctctggtttCCAGAGCAAAA

tctcgat……5'-AGCACNNNNNNNNNNNNNNNNNNNNG3'-GNNNNNNNNNNNNNNNNNNNNCAAAA-5'BsaI5'...GGTCTCNNNNNNN...3'3'...CCAGAGNNNNNNN...5'DoublesgRNABsmAIBsmAI5'...GTCTCNNNNNNN...3'3'...CAGAGNNNNNNN...5'BsmA5'...GTCTCNNNNNNN...3'3'...CAGAGNNNNNNN...5'NNNNNNGAGCAAAAGTCTCNNNNNNNNNNNNCTCTGTTTCAGAGNNNNNNBsmAI……TGATAGAGATACTGAGCACgagcaaaagtctcg

ttttagagctaGAAAtagcaagtt……

…………CTCGTGgctctgtttcagagCAAAA

tctcgat……Combinatorlystyle+85tracrRNAhasmaximalactivity，+67hastheminimalactivityNLS，nuclearlocalizationsignalsgRNAtheguidesequence+tracrmate+tracr+transcriptionterminatorsequenceThelongertrancRNA,thehigherefficiency.(1)N20gttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTT

note:85,83nt?correspondingtopaper,butonemoreTthanthatofthepatent.(2)N20gttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT(67nt)(3)N20gttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT(56nt)

(4)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTTT(NucleicAcidsRes.2013;41(20):e188)ForCas9fromS.pyogenes

SingleguideRNAunderPteto-1promoter:CTCGAGTCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCAC-NNNNNNNNNNNNNNNNNNNN-gttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTTShouldthefirst

Nbethesameasthenaturalbase?PrimershouldcoverthejunctionofcrRNAandpromoter.+1-10-35guidesequencetracrmatesequencetracrsequencetranscriptionalendPLteto-1CloningofmultiplesgRNAsmultiplesgRNAsformultiplexgenomeeditionNucl.AcidsRes.(29October2014)42(19):e147.goldengatecloning：/j5manual/pages/23.htmlMethodsinMolecularBiology，1116,2014,pp119-131MethodsinMolecularBiologyVolume1073,2013,pp141-156goldengatecloningGoldenGateCloningGoldenGateassemblyofsinglelentiviralvectorsencodingCRISPR/Cas9andmultiplesgRNAexpressioncassettes.Inthefirststep,individualsgRNAsareclonedintoseparateexpressionvectorsandsequenceverified.FragmentscontainingeachsgRNAexpressioncassettearethentransferredintoaCas9-expressinglentivirusinstep2byGoldenGateassemblyasshown.BlacktrianglesshowninthelentiviralplasmidrepresentloxPsitesflankingtheentires