Cas9培訓(xùn)技術(shù)講座-2014-09-18

CRISPR/Cas9:生物體和細(xì)胞基因組編輯的革命性技術(shù)南京堯順禹生物科技2014年4月3日敲降〔knockdown〕過表達(dá)〔overexpression)RNA干擾〔RNAi〕MO敲降〔mophorlinoKD〕功能缺失〔過少〕〔lossoffunction)功能獲得〔過多〕〔gainoffunction)全身性過表達(dá)(ubiquitoustransgenic)組織特異性過表達(dá)(tissuespecifictransgenic)基因位點(diǎn)特異性過表達(dá)〔knockin〕全身性敲除〔conventionalKO〕條件性敲除〔conditionalKO〕敲除〔knockout〕基因功能的研究敲降〔RNAi〕2002年Science十大科學(xué)進(jìn)展之首基因敲除(GeneKnockout)基因敲除:將生物體〔或細(xì)胞〕中的基因改變成無功能性基因〔無效等位基因：nullallele〕的一種遺傳學(xué)技術(shù)【Ageneknockout(KO)isagenetictechniqueinwhichoneofanorganism‘sgenesismadeinoperative.】基因打靶(GeneTargeting)基因打靶是利用同源重組原理改變細(xì)胞或生物體內(nèi)源性基因的遺傳學(xué)技術(shù)【Genetargetingisagenetictechniquethatuseshomologousrecombinationtochangeanendogenousgene.】特點(diǎn)〔基因修飾精確、遺傳背景清晰干凈〕：1、刪除基因2、去除外顯子3、定點(diǎn)插入一個基因〔Knockin〕4、引入點(diǎn)突變5、就制作刪除基因或外顯子而言，可實(shí)現(xiàn)永久性(conventional,全身性)或條件性〔conditional,組織或時空〕特異性的基因敲除依賴于胚胎干細(xì)胞的傳統(tǒng)的基因打靶Nextpage百萬分之一的同源重組率；打靶載體含正負(fù)篩選標(biāo)記。78通過將基因敲除的個體或細(xì)胞與正常的個體或細(xì)胞相比較，研究者可以揭示特定基因的功能?；蚯贸夹g(shù)特點(diǎn)：在基因組水平上將目標(biāo)基因修改成無效等位基因，遺傳背景干凈清晰，是研究基因功能的金標(biāo)準(zhǔn)。敲降〔RNAi或MOKD〕的缺點(diǎn)：1、僅局部抑制該基因的功能；2、在轉(zhuǎn)錄后〔包括翻譯〕水平上下調(diào)基因表達(dá)，不能遺傳3、非特異性強(qiáng)；脫靶效率高9MarioRenatoCapecchi

MartinJohnEvans

OliverSmithies

Winnersof2007NobelPrizeinPhysiologyorMedicine

傳統(tǒng)的基因打靶〔GeneTargeting〕的技術(shù)瓶頸1、需要胚胎干細(xì)胞只有小鼠上能建立胚胎干細(xì)胞大鼠的胚胎干細(xì)胞雖有報道，但沒有后續(xù)的工作2、極低的同源重組率干細(xì)胞同源重組發(fā)生率較高，但也只有百萬分之一ESCells–SpeciesLimitationGermlinetransmissionMouseESCells〔innercellmass〕rat？PGCs〔primordialgermcell〕iPS〔inducedpluripotentcells〕Double-strandBreaksandRepairDouble-strandbreaks(DSBs),inwhichbothstrandsinthedoublehelixaresevered,areparticularlyhazardoustothecellbecausetheycanleadtogenomerearrangements.ThreemechanismsexisttorepairDSBs:1)non-homologousendjoining(NHEJ)2)microhomology-mediatedendjoining(MMEJ),3)homologousrecombination(HR)Non-homologousendjoining(NHEJ)NHEJ(coinedin1996byMooreandHaber)isreferredtoas"non-homologous"becausethebreakendsaredirectlyligatedwithouttheneedforahomologoustemplate.Itisaerror-pronerepair.NHEJtypicallyutilizesshorthomologousDNAsequencescalledmicrohomologiestoguiderepair.Thesemicrohomologiesareoftenpresentinsingle-strandedoverhangsontheendsofDSB.Whentheoverhangsareperfectlycompatible,NHEJusuallyrepairsthebreakaccurately.Impreciserepairleadingtolossofnucleotidescanalsooccur,butismuchmorecommonwhentheoverhangsarenotcompatible.InappropriateNHEJcanleadtotranslocationsandtelomerefusion,hallmarksoftumorcells.TheNHEJandHROccurringnearbytheDSBsitesGenetargetingdependsonDSBsitesspontaneousoccurringintargetgenes.IncreasingDSBsitesdefinitelyimprovetheefficiencyofgenetargetingorgeneknockout.MethodstoCreateDSBnearbytheTargetGene

(Restrictionenzyme:anenzymethatcutsDNAatspecificrecognitionnucleotidesequencesknownasrestrictionsites.)劃時代的突破！ZFN技術(shù)Doyonetal,2008.Heritabletargetedgenedisruptioninzebrafishusingdesignedzinc-fingernucleases.NatBiotechnol.2008Jun;26(6):702-8.Epub2008May25.Mengetal,Targetedgeneinactivationinzebrafishusingengineeredzinc-fingernucleases.NatBiotechnol.2008Jun;26(6):695-701

Doyonetal,2008.Heritabletargetedgenedisruptioninzebrafishusingdesignedzinc-fingernucleases.NatBiotechnol.2008Jun;26(6):702-8.Epub2008May25.Mengetal,Targetedgeneinactivationinzebrafishusingengineeredzinc-fingernucleases.NatBiotechnol.2008Jun;26(6):695-701

GenomeEditingUsingZFNTechnologyWhatIsZFNTechnology?Zincfingernucleases(ZFNs)areaclassofengineeredDNA-bindingproteinsthatfacilitatetargetededitingofthegenomebycreatingdouble-strandbreaksinDNAatuser-specifiedlocations.Figure1:EachZincFingerNuclease(ZFN)consistsoftwofunctionaldomains:a.)ADNA-bindingdomaincomprisedofachainoftwo-fingermodules,eachrecognizingauniquehexamer(6bp)sequenceofDNA.Two-fingermodulesarestitchedtogethertoformaZincFingerProtein,eachwithspecificityof≥24bp.b.)ADNA-cleavingdomaincomprisedofthenucleasedomainofFokI.WhentheDNA-bindingandDNA-cleavingdomainsarefusedtogether,ahighly-specificpairof'genomicscissors'arecreated.ZFNKnockoutAnimalCreationviaMicroinjectionZFN：zinc-fingernucleaseDoyonetal,2008.Heritabletargetedgenedisruptioninzebrafishusingdesignedzinc-fingernucleases.NATUREBIOTECHNOLOGYpublishedonline25May2008;doi:10.1038/nbt1409Componentsofyeast-basedchromosomalreportersystem:expressionvectorsfortheZFNs(top),targetgeneintegratedintothechromosomeattheHOlocus(middle).Afteradouble-strandbreak(DSB)isinducedatthetargetbyapairofZFNs,itisprocessedviasingle-strandannealing(SSA)torepairtheMEL1reportergene,theactivityofwhichcanberapidlyassayedinliquidculture.ZFN：zinc-fingernucleaseMengetal,2008.Targetedgeneinactivationinzebrafishusingengineeredzinc-fingernucleases.NATUREBIOTECHNOLOGYpublishedonline25May2008;doi:10.1038/nbt1398Figure1EngineeringZFNsthattargetkdrexon2usingabacterialone-hybridsystem.(a)Firstselectionstagetoidentifysinglezincfingerswithoptimizedbindingtoeach3-bpsubsitewithinarecognitionelement.(b)RecombinationofindividualzincfingersandsubsequentsecondselectionstageagainstfullZFPrecognitionelements.(c)SequencelogosareshownforthebindingspecificityofZFPsincorporatedintoZFNsforsubsequentanalysisinzebrafishembryos.(d)OverviewofapproachforZFN-targetedmutagenesisinzebrafishembryos.24CuiX,JiD,FisherDA,WuY,BrinerDM,etal.(2010)Targetedintegrationinratandmouseembryoswithzinc-fingernucleases.NatBiotechnol29:64-67.ZFN-inducedtargetedintegration(HR)25利用ZFN技術(shù)進(jìn)行基因敲除和基因打靶的技術(shù)瓶頸1、構(gòu)建ZFN很困難，需要進(jìn)行復(fù)雜的分子重組2、獲得有活性的ZFN非常非常困難

研究人員通常還需要驗(yàn)證以及幾十個不同的ZFNs，來證明其中一個有效。articles.view/articleNo/39239/title/A-CRISPR-Fore-Cas-t/26Dongetal.,2011.PLoSONE,6(12):e28897.我們的創(chuàng)造：斑馬魚胚胎作為篩選系統(tǒng)篩選有活性的ZFNHeritabletargetedinactivationofmyostatingeneinyellowcatfish(Pseudobagrusfulvidraco)usingdesignedzincfingernucleasesGenomeEditingUsingTALENTechnologyTAL(transcriptionactivator-like)effectors(TALEs):proteinssecretedbyXanthomonasbacteria(gram-negativebacteria).Theseproteinscanbindpromotersequencesinthehostplantandactivatetheexpressionofplantgenesthataidbacterialinfection.TheyrecognizeplantDNAsequencesthroughacentralrepeatdomainconsistingofavariablenumberof~34aminoacidrepeats.BochJ,BonasU(September2010)."XanthomonasAvrBs3Family-TypeIIIEffectors:DiscoveryandFunction".AnnualReviewofPhytopathology

48:419–36.NucleicAcidsResearch,2011,Vol.39,No.12SCIENCEVOL32611DECEMBER2009UnitAssemblyTALEN技術(shù)靶向突變黃顙魚胚胎細(xì)胞基因組中的mstnbDongetal.,2014.ZebrafishBedellVM,WangY,CampbellJM,PoshustaTL,StarkerCG,etal.(2012)Nature491:114-118.TALEN-inducedtargetedintegration(HR)利用TALEN技術(shù)進(jìn)行基因敲除或基因打靶的技術(shù)瓶頸1、構(gòu)建TALEN依然耗時、耗力2、獲得有活性的TALEN非常困難

研究人員通常還需要驗(yàn)證十幾個不同的TALENS來證明其中一個有效。

基因組工程學(xué)里程碑式的技術(shù)—CRISPR/Cas9Gajetal.,2013.TrendsinBiotechnology,09May2013一、癌癥免疫療法：癌癥治療的標(biāo)靶是身體的免疫系統(tǒng)而不是直接針對腫瘤。二、CRISPR:

這種基因編輯技術(shù)是在細(xì)菌中被發(fā)現(xiàn)的，但研究人員現(xiàn)在將其作為一種外科手術(shù)刀而指向了個體基因。其普及性在今年出現(xiàn)飆升，因?yàn)橛谐^12個研究團(tuán)隊(duì)用它來操控多個植物、動物及人類細(xì)胞的基因組。六、迷你器官:

在體外生長迷你人樣“類器官”上取得了顯著的進(jìn)步。這些類器官包括肝芽、迷你腎及微型大腦。八、人類的克隆胚胎:

成功地從克隆的人類胚胎中得到了干細(xì)胞?！維cience】2014年度十大科學(xué)突破2013年度的10大科學(xué)人物《自然》評選出了2013年度10大科學(xué)人物，張鋒〔FengZhang，音譯〕位列第一。DNA“編輯大師”張鋒CRISPR/Cas9基因組編輯技術(shù)CRISPR：(ClusteredRegularlyInterspacedShortPalindromicRepeats)arelocicontainingmultipleshortdirectrepeatsthatarefoundinthegenomesofabout40%ofsequencedbacteriaand90%ofsequencedarchaea.IshinoY,ShinagawaH,MakinoK,AmemuraM,NakataA(1987)."Nucleotidesequenceoftheiapgene,responsibleforalkalinephosphataseisozymeconversioninEscherichiacoli,andidentificationofthegeneproduct".JBacteriol169(12):5429–33.RepeatMojica,F.J.;Díez-Villase?or,C.;Soria,E.;Juez,G.(2000)."BiologicalsignificanceofafamilyofregularlyspacedrepeatsinthegenomesofArchaea,Bacteriaandmitochondria".Molecularmicrobiology36(1):244–246.doi:10.1046/j.1365-2958.2000.01838.x.SRSRJansen,R.;Embden,J.D.;Gaastra,W.;Schouls,L.M.(2002)."IdentificationofgenesthatareassociatedwithDNArepeatsinprokaryotes".Molecularmicrobiology43(6):1565–1575.CRISPR

HorvathP,BarrangouR(January2010)."CRISPR/Cas,theimmunesystemofbacteriaandarchaea".Science327(5962):167–70.

ShortsegmentsofforeignDNA,calledspacers,areincorporatedintothegenomebetweenCRISPRrepeats,andserveasa'memory'ofpastexposures.CRISPRspacersarethenusedtorecognizeandsilenceexogenousgeneticelementsinamanneranalogoustoRNAiineukaryoticorganisms.CRISPRFunctionsasaProkaryoticImmuneSystem(acquiredimmunity)Milestonework:JinekM,ChylinskiK,FonfaraI,HauerM,DoudnaJA,CharpentierEAprogrammabledual-RNA-guidedDNAendonucleaseinadaptivebacterialimmunity.Science.2012Aug17;337(6096):816-21.

Cas9、tracrRNA和crRNA一起以某種方法攻擊和crRNA配對的外來DNA

李君等,2013.CRISPR/Cas系統(tǒng)：RNA靶向的基因組定向編輯新技術(shù)遺傳

35(11):1265―1273Cas9是一個核酸內(nèi)切酶Cas9是由1409個aa組成〔加上兩端的核定位信號，編碼序列全長4.5kb〕含有2個核酸內(nèi)切酶結(jié)構(gòu)域：中間位置的HNH核酸內(nèi)切酶結(jié)構(gòu)域〔H結(jié)構(gòu)域，切割與crRNA互補(bǔ)的鏈〕氨基端RuvC-like〔R結(jié)構(gòu)域，切割另外一條鏈〕CRISPR/Cas9識別目標(biāo)基因組DNA示意圖(改自Malietal.,2013.Science,339(6121):823-6.)SchematicdiagramofCRISPR/Cas9systemCas9適用的應(yīng)用研究：敲除應(yīng)用于動植物及微生物基因組水平上的編碼基因的靶向基因敲除應(yīng)用于動植物基因組水平上的非編碼基因如lncRNA〔longnon-codingRNA〕和microRNA的靶向基因敲除多基因同時敲除47Cas9適用的應(yīng)用研究：敲入應(yīng)用于動植物及微生物基因組水平上的編碼基因的靶向基因敲入點(diǎn)突變、保守區(qū)域替換、或參加標(biāo)簽編碼序列李君等,2013.CRISPR/Cas系統(tǒng)：RNA靶向的基因組定向編輯新技術(shù)遺傳

35(11):1265―1273Genome-scaleScreeningGenome-scaleCRISPR-Cas9knockoutscreeninginhumancells.ShalemO,SanjanaNE,HartenianE,ShiX,ScottDA,MikkelsenTS,HecklD,EbertBL,RootDE,DoenchJG,ZhangF.Science.2014Jan3;343(6166):84-7.doi:10.1126/science.1247005.Epub2013Dec12.GeneticscreensinhumancellsusingtheCRISPR-Cas9system.WangT,WeiJJ,SabatiniDM,LanderES.Science.2014Jan3;343(6166):80-4.doi:10.1126/science.1246981.Epub2013Dec12.Cas9適用的應(yīng)用研究：其它領(lǐng)域雙突變的Cas9〔dCas9〕的C端加上VP16等具激活活性的功能結(jié)構(gòu)域，那么可以特異地激活目標(biāo)基因的表達(dá)在雙突變的Cas9〔dCas9〕的C端加上EVE等抑制活性的功能結(jié)構(gòu)域，那么可以特異地抑制目標(biāo)基因的表達(dá)〔Cell.2013Jul18;154(2):442-51〕。應(yīng)用于動植物基因組水平上目標(biāo)基因的甲基化、去甲基化等表觀修飾。Cas9實(shí)驗(yàn)的常見問題：脫靶問題脫靶幾率計算：如果目標(biāo)位點(diǎn)突變率為30%，另外潛在位點(diǎn)的突變率〔脫靶率〕1%，因?yàn)檫@兩個事件是獨(dú)立事件，因此在指定的細(xì)胞中，既發(fā)生目標(biāo)位點(diǎn)基因敲除同時又發(fā)生脫靶的概率即為30%1%=0.3%，是非常低的。是可控的。在一般的以研究為目的的實(shí)驗(yàn)中，脫靶并不是問題，只有用于治療才是個問題RNAi的敲降也一樣存在脫靶問題，只不過之前沒有重視過。。。。。Talen也存在，只是做的人更多偏向Cas9技術(shù)OffTargetproblemCRISPR/Cas9技術(shù)脫靶問題已獲解決17個堿基的設(shè)計GNNNNN。。NGG，比常規(guī)降低了5000倍，幾乎就不再脫靶。17個再加上雙切口技術(shù)，脫靶幾乎檢測不到！SolvedOffTargetproblem：Cas9〔D10A〕介導(dǎo)的雙切口增強(qiáng)基因組編輯的特異性SolvedOffTargetproblem：使用17nt或18ntgRNA增強(qiáng)Cas9對基因組編輯的特異性最大限度地解決脫靶問題：17ntgRNA+

Cas9n介導(dǎo)的雙切口可最大程度地提高基因組編輯的特異性FuandSander,2014,InpressRanetal.,2013Sep12;154(6):1380-9文章案例EffectivegenetargetinginrabbitsusingRNA-guidedCas9nucleasesdoi:10.1093/jmcb/mjt04150ng/ulCas9mRNAplus6ng/ulsgRNARNA-guidedendonucleasesHighlyefficientgeneknockoutinmiceandzebrafishwithRNA-guidedendonucleasesHyun-TaekKimpublishedonlineNovember19,2013GenomeResOne-StepGenerationofMiceCarryingMutationsinMultipleGenesbyCRISPR/Cas-MediatedGenomeEngineeringRudolfJaenischCell153,910–918,May9,2013GenerationofGene-ModifiedCynomolgusMonkeyviaCas9/RNA-MediatedGeneTargetinginOne-CellEmbryosCell156,1–8,February13,2014Cas9mRNA(20ng/ml)andsgRNAs(5ng/ml)為什么Cas9是最受關(guān)注的？原因：構(gòu)建簡單，guideRNA只需17-23個效率：RNA-DNA>蛋白-DNA可高通量：一次性敲除多個基因Cas9細(xì)胞怎么做確定待敲除基因的靶位點(diǎn)并設(shè)計識別靶位點(diǎn)的識別的一對DNAOligo〔引物〕2.構(gòu)建可表達(dá)sgRNA的Cas9質(zhì)粒3.含sgRNA表達(dá)元件的Cas9質(zhì)粒的活性檢測4.利用Cas9質(zhì)粒建立knock-out細(xì)胞系1，如何設(shè)計？以CFTR為例NCBI中找到對應(yīng)的exon2構(gòu)建可表達(dá)sgRNA的Cas9質(zhì)粒

將合成的Oligos以逐步降溫的方法退火成雙鏈，然后與堯順禹生物提供的Cas9質(zhì)粒進(jìn)行連接，連接后轉(zhuǎn)化感受態(tài)的大腸桿菌，再進(jìn)行涂板。YSY-CRISPR/Cas9Kit將正反向兩個引物進(jìn)行退火將退火產(chǎn)物與線性化的質(zhì)粒連接轉(zhuǎn)化感受態(tài)細(xì)胞轉(zhuǎn)化子鑒定送2個經(jīng)PCR驗(yàn)證有陽性條帶的菌液去測序使用質(zhì)粒提取試劑盒從經(jīng)測序確認(rèn)含正確重組質(zhì)粒的菌液中提取質(zhì)粒。電轉(zhuǎn)如293T、3T3等模式細(xì)胞或目的細(xì)胞脂轉(zhuǎn)熒光鑒定轉(zhuǎn)染效率Puro藥篩3-5天局部細(xì)胞抽提DNA、并PCR送測序通過測序套峰情況初步判斷活性效率PCR產(chǎn)物連接TA克隆，驗(yàn)證準(zhǔn)確效率3含sgRNA表達(dá)元件的Cas9質(zhì)粒的活性檢測

YSYsgRNA活性檢測〔專利〕含識別位點(diǎn)的基因組片段〔400-600bp〕gRNA+Cas9mRNA斑馬魚受精卵發(fā)育24hpfYSY超微量試劑盒5個胚胎混合制備DNA模板通過測序套峰情況初步判斷活性效率PCR產(chǎn)物連接TA克隆，驗(yàn)證準(zhǔn)確效率套峰圖4.利用Cas9質(zhì)粒建立knock-out細(xì)胞系

電轉(zhuǎn)

目的細(xì)胞脂轉(zhuǎn)熒光鑒定轉(zhuǎn)染效率Puro藥篩3-5天其余細(xì)胞消化成單細(xì)胞克隆，繼續(xù)培養(yǎng)并結(jié)合效率數(shù)據(jù)篩選獲得穩(wěn)定敲除細(xì)胞系PCR檢測單克隆細(xì)胞，篩選非3的整數(shù)倍突變穩(wěn)定建系成功Cas9動物模