基因编辑技术及其在中国的研究发展

遗传,2018,40(10):900-915doi:10.16288/j.yczz.18-195

综述

2.北京大学生物医学前沿创新中心,北京100871

3.北京未来基因诊断高精尖创新中心,北京100871

4.北大-清华生命科学联合中心,北京100871

5.北京大学前沿交叉学科研究院,北京100871

6.北京大学基因组编辑研究中心,北京100871

2.BiomedicalPioneeringInnovationCenter,PekingUniversity,Beijing100871,China

3.BeijingAdvancedInnovationCenterofGenomics,Beijing100871,China

4.PKU-TsinghuaCenterforLifeSciences,Beijing100871,China

5.AcademyforAdvancedInterdisciplinaryStudies,PekingUniversity,Beijing100871,China

6.PekingUniversityGenomeEditingResearchCenter,Beijing100871,China

编委:高彩霞

收稿日期:2018-07-10修回日期:2018-09-7网络出版日期:2018-10-20

Received:2018-07-10Revised:2018-09-7Online:2018-10-20

作者简介Aboutauthors

基因编辑技术是一种能够对生物体的基因组及其转录产物进行定点修饰或者修改的技术,早期基因编辑技术包括归巢内切酶、锌指核酸内切酶和类转录激活因子效应物。近年来,以CRISPR/Cas9系统为代表的新型技术使基因编辑的研究和应用领域得以迅速拓展。本文对基因编辑技术的原理、技术发展及其应用进行了阐述,对我国在基因编辑机制研究及技术发展、基因编辑动植物模型构建、基因治疗等领域的研究进展进行了回顾,并对基因技术的发展前景及趋势进行了展望。

关键词:基因编辑;遗传工程;CRISPR;基因功能;疾病模型;基因治疗

Geneeditingisatechnologyforprecisionmodificationofgenomesequencesorexpressingtranscripts.Earlyversionsofsuchtechniquesincludehomingendonuclease,zincfingerendonucleaseandtranscription-likeactivatoreffectors.TheCRISPR/Cas9systemhasrecentlyemergedasapowerfulandversatilegeneeditingtoolforabroadspectrumofapplications.Inthisreview,wediscusstherecentdevelopmentandapplicationsofgeneeditingtechnology,andparticularlyfocusontheprogressinChina,withtheemphasisonmechanisticstudies,developmentofnewandextendedtechnologies,theapplicationsinanimals,plantsandthetherapeutics.

Keywords:geneediting;geneticengineering;CRISPR;genefunction;diseasemodel;genetherapy

本文引用格式

陈一欧,宝颖,马华峥,伊宗裔,周卓,魏文胜.基因编辑技术及其在中国的研究发展[J].遗传,2018,40(10):900-915doi:10.16288/j.yczz.18-195

YiouChen,YingBao,HuazhengMa,ZongyiYi,ZhuoZhou,WenshengWei.GeneeditingtechnologyanditsresearchprogressinChina[J].Hereditas(Beijing),2018,40(10):900-915doi:10.16288/j.yczz.18-195

早期基因编辑技术包括归巢内切酶(homingendonuclease,HEs)、锌指核酸内切酶(zincfingerendonuclease,ZFN)和类转录激活因子效应物(transcriptionactivator-likeeffectornucleases,TALENs),但脱靶效应或组装复杂性限制了这些技术在基因编辑领域中的应用。近年来,以CRISPR/Cas9系统为代表的新型基因编辑技术飞速发展,并开始在诸多生物学领域中得到广泛应用。

1.1.1归巢内切酶

1.1.2ZFNs和TALENs技术

1.2.1CRISPR/Cas9系统的发现

1.2.2CRISPR/Cas9系统的作用机制

1.2.3CRISPR/Cas9系统的发展

近年来,我国科学家在基因编辑领域取得了令人鼓舞的进展,在基因编辑系统发展、机制研究、构建基因编辑动植物模型和基因治疗等方面取得了突出的成绩。

图2李家洋在中国遗传学会基因组编辑分会成立大会上发言

Fig.2JiayangLispokeattheinauguralmeetingofthegenomeeditingbranchofthegeneticssocietyofChina

对CRISPR/Cas9系统的优化包括探索未知的CRISPR家族蛋白外以及对已知的Cas蛋白进行改造。针对大家较为熟知的Cas9蛋白,改造主要包括以下3个方面。

3.1.1减少Cas9蛋白体积,使其更易于通过病毒载体系统进行体内表达

3.1.2减少Cas9蛋白脱靶效应,提高专一性和保真度

3.1.3增加Cas9蛋白的靶向范围,即拓展PAM识别范围

3.2.1碱基编辑系统

3.2.2CRISPR介导的基因表达调控

基因编辑技术在基因功能研究、药物开发、疾病治疗和作物育种等方面有着重要意义和广阔的应用前景。如前文所述,基因编辑技术可以在全基因组范围进行基因功能研究,除此之外,该技术也被广泛应用于生物治疗以及药物研究等领域。

很多疾病的致病机制十分复杂,比如癌症,通常涉及多种抑癌基因或致癌基因的遗传改变。因此构建适合的疾病模型对探索疾病的发生和进展以及抗癌药物的筛选有着重要的意义。对于已知致病基因的疾病,研究人员可以运用CRISPR/Cas9等基因编辑技术,构建对应的基因突变动物或细胞模型,从而进一步进行药物或其他治疗方式的研究。对于功能未知或者部分未知的基因,研究人员可以通过构建疾病模型从而进一步明确疾病与基因之间的关系。

随着研究者的不断深入探索,基因编辑技术正在逐步发展成熟,具有极大的研究潜力和广阔的应用前景。相信在不久的将来,基因编辑技术会在人类生产和临床疾病治疗中发挥其无可替代的价值。

(责任编委:高彩霞)

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Koike-YusaH,LiY,TanEP,Velasco-HerreraMdelC,YusaK.Genome-widerecessivegeneticscreeninginmammaliancellswithalentiviralCRISPR-guideRNAlibrary

WangT,WeiJJ,SabatiniDM,LanderES.GeneticscreensinhumancellsusingtheCRISPR-Cas9system

ChenJS,MaE,HarringtonLB,DaCostaM,TianX,PalefskyJM,DoudnaJA.CRISPR-Cas12atargetbindingunleashesindiscriminatesingle-strandedDNaseactivity

CRISPR-Cas12a(Cpf1)proteinsareRNA-guidedenzymesthatbindandcutDNAascomponentsofbacterialadaptiveimmunesystems.LikeCRISPR-Cas9,Cas12ahasbeenharnessedforgenomeeditingonthebasisofitsabilitytogeneratetargeted,double-strandedDNAbreaks.HereweshowthatRNA-guidedDNAbindingunleashesindiscriminatesingle-strandedDNA(ssDNA)cleavageactivitybyCas12athatcompletelydegradesssDNAmolecules.Wefindthattarget-activated,nonspecificsingle-strandeddeoxyribonuclease(ssDNase)cleavageisalsoapropertyofothertypeVCRISPR-Cas12enzymes.BycombiningCas12assDNaseactivationwithisothermalamplification,wecreateamethodtermedDNAendonuclease-targetedCRISPRtransreporter(DETECTR),whichachievesattomolarsensitivityforDNAdetection.DETECTRenablesrapidandspecificdetectionofhumanpapillomavirusinpatientsamples,therebyprovidingasimpleplatformformoleculardiagnostics.

QiLS,LarsonMH,GilbertLA,DoudnaJA,WeissmanJS,ArkinAP,LimWA.RepurposingCRISPRasanRNA-Guidedplatformforsequence-specificcontrolofgeneexpression

TheauthorshavedevelopedaCRISPRinterferencesysteminwhichacatalyticallydeadCas9proteincanbetargetedtoaspecificgenomicsitethroughacomplementarysmallguideRNA,allowingsystematicperturbationofgenetranscriptioninbacteriaandmammaliancells.

HuJH,MillerSM,GeurtsMH,TangW,ChenL,SunN,ZeinaCM,GaoX,ReesHA,LinZ,LiuDR.EvolvedCas9variantswithbroadPAMcompatibilityandhighDNAspecificity

ProgrammableDNAnucleaseshaveprovidedscientistswiththeunprecedentedabilitytoprobe,regulate,andmanipulatethehumangenome.Zinc-fingernucleases(ZFNs),transcriptionactivator-likeeffectornucleases(TALENs),andtheclusteredregularlyinterspacedshortpalindromicrepeat-Cas9system(CRISPR-Cas9)representapowerfularrayoftoolsthatcanbindtoandcleaveaspecifiedDNA...[Showfullabstract]

FonfaraI,RichterH,BratovicM,LeRhunA,CharpentierE.TheCRISPR-associatedDNA-cleavingenzymeCpf1alsoprocessesprecursorCRISPRRNA

YamanoT,NishimasuH,ZetscheB,HiranoH,SlaymakerIM,LiYQ,FedorovaI,NakaneT,MakarovaKS,KooninEV,IshitaniR,ZhangF,NurekiO.CrystalstructureofCpf1incomplexwithguideRNAandtargetDNA

ChenJS,MaEB,HarringtonLB,DaCostaM,TianXR,PalefskyJM,DoudnaJA.CRISPR-Cas12atargetbindingunleashesindiscriminatesingle-strandedDNaseactivity

LiSY,ChengQX,LiuJK,NieXQ,ZhaoGP,WangJ.CRISPR-Cas12ahasbothcis-andtrans-cleavageactivitiesonsingle-strandedDNA

Developnewtools

ZetscheB,HeidenreichM,MohanrajuP,FedorovaI,KneppersJ,DegennaroEM,WinbladN,ChoudhurySR,AbudayyehO,GootenbergJS,WuWY,ScottDA,SeverinovK,VanDerOostJ,ZhangF.MultiplexgeneeditingbyCRISPR-Cpf1usingasinglecrRNAarray

TargetingofmultiplegenomiclociwithCas9islimitedbytheneedformultipleorlargeexpressionconstructs.HereweshowthattheabilityofCpf1toprocessitsownCRISPRRNA(crRNA)canbeusedtosimplifymultiplexedgenomeediting.UsingasinglecustomizedCRISPRarray,weedituptofourgenesinmammaliancellsandthreeinthemousebrain,simultaneously.

WangMG,MaoYF,LuYM,TaoXP,ZhuJK.MultiplexgeneeditinginriceusingtheCRISPR-Cpf1system

LiXS,WangY,LiuYJ,YangB,WangX,WeiJ,LuZY,ZhangYX,WuJ,HuangXX,YangL,ChenJ.BaseeditingwithaCpf1-cytidinedeaminasefusion

TakYE,KleinstiverBP,NunezJK,HsuJY,HorngJE,GongJY,WeissmanJS,JoungJK.InducibleandmultiplexgeneregulationusingCRISPR-Cpf1-basedtranscriptionfactors

AbstractTargetedandinducibleregulationofmammaliangeneexpressionisabroadlyimportantcapability.Weengineereddrug-induciblecatalyticallyinactiveCpf1nucleasefusedtotranscriptionalactivationdomainstotunetheexpressionofendogenousgenesinhumancells.LeveragingthemultiplexcapabilityoftheCpf1platform,wedemonstratebothsynergisticandcombinatorialgeneexpressioninhumancells.Ourworkshouldenablethedevelopmentofmultiplexgeneperturbationlibraryscreensforunderstandingcomplexcellularphenotypes.

GaoLY,CoxDBT,YanWX,ManteigaJC,SchneiderMW,YamanoT,NishimasuH,NurekiO,CrosettoN,ZhangF.EngineeredCpf1variantswithalteredPAMspecificities

LiSY,ZhangX,WangWS,GuoXP,WuZC,DuWM,ZhaoYD,XiaLQ.ExpandingthescopeofCRISPR/Cpf1-mediatedgenomeeditinginrice

Cerealshighinamylosecontent(AC)andresistantstarch(RS)offerpotentialhealthbenefits.PreviousstudiesusingchemicalmutagenesisorRNAinterferencehavedemonstratedthatstarchbranchingenzyme(SBE)playsamajorroleindeterminingthefinestructureandphysicalpropertiesofstarch.However,itremainsachallengetocontrolstarchbranchingincommerciallines.Here,weuse...[Showfullabstract]

GuoM,WangS,ZhuY,WangS,XiongZ,YangJ,XuZ,HuangZ.StructuralbasisofCRISPR-SpyCas9inhibitionbyananti-CRISPRprotein

DongD,RenK,QiuX,ZhengJ,GuoM,GuanX,LiuH,LiN,ZhangB,YangD,MaC,WangS,WuD,MaY,FanS,WangJ,GaoN,HuangZ.ThecrystalstructureofCpf1incomplexwithCRISPRRNA

LiuL,LiX,MaJ,LiZ,YouL,WangJ,WangM,ZhangX,WangY.ThemoleculararchitectureforRNA-guidedRNAcleavagebyCas13a

Cas13a,atypeVI-ACRISPR-CasRNA-guidedRNAribonuclease,degradesinvasiveRNAstargetedbyCRISPRRNA(crRNA)andhaspotentialapplicationsinRNAtechnology.TounderstandhowCas13aisactivatedtocleaveRNA,wehavedeterminedthecrystalstructureofLeptotrichiabuccalis(Lbu)Cas13aboundtocrRNAanditstargetRNA,aswellasthecryo-EMstructureoftheLbuCas13a-crRNAcomplex.ThecrRNA-targetRNAduplexbindsinapositivelychargedcentralchannelofthenuclease(NUC)lobe,andCas13aproteinandcrRNAundergoasignificantconformationalchangeupontargetRNAbinding.Theguide-targetRNAduplexformationtriggersHEPN1domaintomovetowardHEPN2domain,activatingtheHEPNcatalyticsiteofCas13aprotein,whichsubsequentlycleavesbothsingle-strandedtargetandcollateralRNAsinanon-specificmanner.ThesefindingsrevealhowCas13aoftypeVICRISPR-CassystemsdefendagainstRNAphagesandsetthestageforitsdevelopmentasatoolforRNAmanipulation.

LiuL,LiX,WangJ,WangM,ChenP,YinM,LiJ,ShengG,WangY.TwodistantcatalyticsitesareresponsibleforC2c2RNaseactivities

C2c2,theeffectoroftypeVICRISPR-Cassystems,hastwoRNaseactivitiesneforcuttingitsRNAtargetandtheotherforprocessingtheCRISPRRNA(crRNA).Here,wereportthestructuresofLeptotrichiashahiiC2c2initscrRNA-freeandcrRNA-boundstates.WhileC2c2hasabilobedstructurereminiscentofallotherClass2effectors,italsoexhibitsdifferentstructuralcharacteristics.ItcontainstheREClobewithaHelical-1domainandtheNUClobewithtwoHEPNdomains.ThetwoRNasecatalyticpocketsresponsibleforcleavingpre-crRNAandtargetRNAareindependentlylocatedonHelical-1andHEPNdomains,respectively.crRNAbindinginducessignificantconformationalchangesthatarelikelytostabilizecrRNAbindingandfacilitatetargetRNArecognition.ThesestructuresprovideimportantinsightsintothemolecularmechanismofdualRNaseactivitiesofC2c2andestablishaframeworkforitsfutureengineeringasaRNAeditingtool.

WangJ,LiJ,ZhaoH,ShengG,WangM,YinM,WangY.StructuralandmechanisticbasisofPAM-dependentspaceracquisitioninCRISPR-Cassystems

MaY,ZhangJ,YinW,ZhangZ,SongY,ChangX.TargetedAID-mediatedmutagenesis(TAM)enablesefficientgenomicdiversificationinmammaliancells

LiX,WangY,LiuY,YangB,WangX,WeiJ,LuZ,ZhangY,WuJ,HuangX,YangL,ChenJ.BaseeditingwithaCpf1-cytidinedeaminasefusion

ZhuS,LiW,LiuJ,ChenCH,LiaoQ,XuP,XuH,XiaoT,CaoZ,PengJ,YuanP,BrownM,LiuXS,WeiW.Genome-scaledeletionscreeningofhumanlongnon-codingRNAsusingapaired-guideRNACRISPR-Cas9library

LiW,TengF,LiT,ZhouQ.SimultaneousgenerationandgermlinetransmissionofmultiplegenemutationsinratusingCRISPR-Cassystems

YanS,TuZ,LiuZ,FanN,YangH,YangS,YangW,ZhaoY,OuyangZ,LaiC,YangH,LiL,LiuQ,ShiH,XuG,ZhaoH,WeiH,PeiZ,LiS,LaiL,LiXJ.AhuntingtinknockinpigmodelrecapitulatesfeaturesofselectiveneurodegenerationinHuntington'sdisease

KeQ,LiW,LaiX,ChenH,HuangL,KangZ,LiK,RenJ,LinX,ZhengH,HuangW,MaY,XuD,ChenZ,SongX,LinX,ZhuangM,WangT,ZhuangF,XiJ,MaoFF,XiaH,LahnBT,ZhouQ,YangS,XiangAP.TALEN-basedgenerationofacynomolgusmonkeydiseasemodelforhumanmicrocephaly

Geneeditinginnon-humanprimatesmayleadtovaluablemodelsforexploringtheetiologiesandtherapeuticstrategiesofgeneticallybasedneurologicaldisordersinhumans.However,amonkeymodelofneurologicaldisordersthatcloselymimicspathologicalandbehavioraldeficitsinhumanshasnotyetbeensuccessfullygenerated.Microcephalin1(MCPH1)isimplicatedintheevolutionofthehumanbrain,andMCPH1mutationcausesmicrocephalyaccompaniedbymentalretardation.Herewegeneratedacynomolgusmonkey(Macacafascicularis)carryingbiallelicMCPH1mutationsusingtranscriptionactivator-likeeffectornucleases.Themonkeyrecapitulatedmostoftheimportantclinicalfeaturesobservedinpatients,includingmarkedreductionsinheadcircumference,prematurechromosomecondensation(PCC),hypoplasiaofthecorpuscallosumandupperlimbspasticity.Moreover,overexpressionofMCPH1inmutateddermalfibroblastsrescuedthePCCsyndrome.ThismonkeymodelmayhelpuselucidatetheroleofMCPH1inthepathogenesisofhumanmicrocephalyandbetterunderstandthefunctionofthisproteinintheevolutionofprimatebrainsize.

ChenY,YuJ,NiuY,QinD,LiuH,LiG,HuY,WangJ,LuY,KangY,JiangY,WuK,LiS,WeiJ,HeJ,WangJ,LiuX,LuoY,SiC,BaiR,ZhangK,LiuJ,HuangS,ChenZ,WangS,ChenX,BaoX,ZhangQ,LiF,GengR,LiangA,ShenD,JiangT,HuX,MaY,JiW,SunYE.ModelingRettsyndromeusingTALEN-editedMECP2mutantcynomolgusmonkeys

SubjectCode:H09WiththesupportbytheNationalNaturalScienceFoundationofChina,acollaborativestudybytheresearchgroupledbyProf.ChenYongchang(陈永昌)andJiWeizhifromtheYunnanKeyLaboratoryofPrimateBiomedicineResearch&InstituteofPrimateTranslationalMedicine,KunmingUniversityof

YaoX,LiuZ,WangX,WangY,NieYH,LaiL,SunR,ShiL,SunQ,YangH.Generationofknock-incynomolgusmonkeyviaCRISPR/Cas9editing

Knock-inmicegeneratedbyHMEJ-mediatedtargetedintegration

CuiY,NiuY,ZhouJ,ChenY,ChengY,LiS,AiZ,ChuC,WangH,ZhengB,ChenX,ShaJ,GuoX,HuangX,JiW.GenerationofapreciseOct4-hrGFPknockincynomolgusmonkeymodelviaCRISPR/Cas9-assistedhomologousrecombination

FengZ,ZhangB,DingW,LiuX,YangDL,WeiP,CaoF,ZhuS,ZhangF,MaoY,ZhuJK.EfficientgenomeeditinginplantsusingaCRISPR/Cassystem

ZongY,WangY,LiC,ZhangR,ChenK,RanY,QiuJL,WangD,GaoC.Precisebaseeditinginrice,wheatandmaizewithaCas9-cytidinedeaminasefusion

SingleDNAbasepairsareeditedinwheat,riceandmaizeusingaCas9nickasefusionprotein.

WangY,ChengX,ShanQ,ZhangY,LiuJ,GaoC,QiuJL.Simultaneouseditingofthreehomoeoallelesinhexaploidbreadwheatconfersheritableresistancetopowderymildew

ShanQ,WangY,LiJ,ZhangY,ChenK,LiangZ,ZhangK,LiuJ,XiJJ,QiuJL,GaoC.TargetedgenomemodificationofcropplantsusingaCRISPR-Cassystem

ThearticleoffersinformationongenomemodificationofcropplantsusingaCRISPR-Cassystem.Itstatesthatgenomeeditingtechnologiesusingzincfingernucleases(ZFNs)andtranscriptionactivator-likeeffectornucleases(TALENs)canalsogenerategenomemodifications.PhotographsrelatedtogenomeeditinginriceandwheatusinganengineeredtypeIICRISPR-Cassystemarealsopresented.

MaX,ZhangQ,ZhuQ,LiuW,ChenY,QiuR,WangB,YangZ,LiH,LinY,XieY,ShenR,ChenS,WangZ,ChenY,GuoJ,ChenL,ZhaoX,DongZ,LiuYG.ArobustCRISPR/Cas9systemforconvenient,high-efficiencymultiplexgenomeeditinginmonocotanddicotplants

CyranoskiD.ChinesescientiststopioneerfirsthumanCRISPRtrial

Gene-editingtechniquetotreatlungcancerisduetobetestedinpeopleinAugust.

KimE,KooT,ParkSW,KimD,KimK,ChoHY,SongDW,LeeKJ,JungMH,KimS,KimJH,KimJH,KimJS.InvivogenomeeditingwithasmallCas9orthologuederivedfromCampylobacterjejuni

TsaiSQ,ZhengZ,NguyenNT,LiebersM,TopkarVV,ThaparV,WyvekensN,KhayterC,IafrateAJ,LeLP,AryeeMJ,JoungJK.GUIDE-seqenablesgenome-wideprofilingofoff-targetcleavagebyCRISPR-Casnucleases

KimD,BaeS,ParkJ,KimE,KimS,YuHR,HwangJ,KimJI,KimJS.Digenome-seq:genome-wideprofilingofCRISPR-Cas9off-targeteffectsinhumancells

AbstractAlthoughRNA-guidedgenomeeditingviatheCRISPR-Cas9systemisnowwidelyusedinbiomedicalresearch,genome-widetargetspecificitiesofCas9nucleasesremaincontroversial.HerewepresentDigenome-seq,invitroCas9-digestedwhole-genomesequencing,toprofilegenome-wideCas9off-targeteffectsinhumancells.Thisinvitrodigestyieldssequencereadswiththesame5'endsatcleavagesitesthatcanbecomputationallyidentified.Wevalidatedoff-targetsitesatwhichinsertionsordeletionswereinducedwithfrequenciesbelow0.1%,nearthedetectionlimitoftargeteddeepsequencing.WealsoshowedthatCas9nucleasescanbehighlyspecific,inducingoff-targetmutationsatmerelyseveral,ratherthanthousandsof,sitesintheentiregenomeandthatCas9off-targeteffectscanbeavoidedbyreplacing'promiscuous'singleguideRNAs(sgRNAs)withmodifiedsgRNAs.Digenome-seqisarobust,sensitive,unbiasedandcost-effectivemethodforprofilinggenome-wideoff-targeteffectsofprogrammablenucleasesincludingCas9.

SlaymakerIM,GaoL,ZetscheB,ScottDA,YanWX,ZhangF.RationallyengineeredCas9nucleaseswithimprovedspecificity

KleinstiverBP,PattanayakV,PrewMS,TsaiSQ,NguyenNT,ZhengZ,JoungJK.High-fidelityCRISPR-Cas9nucleaseswithnodetectablegenome-wideoff-targeteffects

ChenJS,DagdasYS,KleinstiverBP,WelchMM,SousaAA,HarringtonLB,SternbergSH,JoungJK,YildizA,DoudnaJA.EnhancedproofreadinggovernsCRISPR-Cas9targetingaccuracy

TheRNA-guidedCRISPR-Cas9nucleasefromStreptococcuspyogenes(SpCas9)hasbeenwidelyrepurposedforgenomeediting1–4.High-fidelity(SpCas9-HF1)andenhancedspecificity(eSpCas9(1.1))variantsexhibitsubstantiallyreducedoff-targetcleavageinhumancells,butthemechanismoftargetdiscriminationandthepotentialtofurtherimprovefidelitywereunknown5–9.Usingsingle-moleculeF02rsterresonanceenergytransfer(smFRET)experiments,weshowthatbothSpCas9-HF1andeSpCas9(1.1)aretrappedinaninactivestate10whenboundtomismatchedtargets.Wefindthatanon-catalyticdomainwithinCas9,REC3,recognizestargetcomplementarityandgovernstheHNHnucleasetoregulateoverallcatalyticcompetence.Exploitingthisobservation,wedesignedanewhyper-accurateCas9variant(HypaCas9)thatdemonstrateshighgenome-widespecificitywithoutcompromisingon-targetactivityinhumancells.Theseresultsofferamorecomprehensivemodeltorationalizeandmodifythebalancebetweentargetrecognitionandnucleaseactivationforprecisiongenomeediting.

LinS,StaahlBT,AllaRK,DoudnaJA.Enhancedhomology-directedhumangenomeengineeringbycontrolledtimingofCRISPR/Cas9delivery

KimS,KimD,ChoSW,KimJ,KimJS.HighlyefficientRNA-guidedgenomeeditinginhumancellsviadeliveryofpurifiedCas9ribonucleoproteins

KleinstiverBP,PrewMS,TsaiSQ,TopkarVV,NguyenNT,ZhengZ,GonzalesAP,LiZ,PetersonRT,YehJR,AryeeMJ,JoungJK.EngineeredCRISPR-Cas9nucleaseswithalteredPAMspecificities

EngineeredCRISPR-Cas9nucleaseswithalteredandimprovedPAMspecificitiesandtheiruseingenomicengineering,epigenomicengineering,andgenometargeting.

KleinstiverBP,PrewMS,TsaiSQ,NguyenNT,TopkarVV,ZhengZ,JoungJK.BroadeningthetargetingrangeofStaphylococcusaureusCRISPR-Cas9bymodifyingPAMrecognition

HiranoH,GootenbergJS,HoriiT,AbudayyehOO,KimuraM,HsuPD,NakaneT,IshitaniR,HatadaI,ZhangF,NishimasuH,NurekiO.StructureandengineeringofFrancisellanovicidaCas9

AdliM.TheCRISPRtoolkitforgenomeeditingandbeyond

Autoimmunediseasesareenigmaticandcomplex,andmostbeenassociatedwithepigeneticchanges.Epigeneticsdescribeschangesingeneexpressionrelatedtoenvironmentalinfluencesmediatedbyavarietyofeffectorsthatalterthethree-dimensionalstructureofchromatinandfacilitatetranscriptionfactororrepressorbinding.Recentyearshavewitnessedadramaticchangeandaccelerationin...[Showfullabstract]

KomorAC,KimYB,PackerMS,ZurisJA,LiuDR.ProgrammableeditingofatargetbaseingenomicDNAwithoutdouble-strandedDNAcleavage

GaudelliNM,KomorAC,ReesHA,PackerMS,BadranAH,BrysonDI,LiuDR.ProgrammablebaseeditingofA*TtoG*CingenomicDNAwithoutDNAcleavage

HessGT,FresardL,HanK,LeeCH,LiA,CimprichKA,MontgomerySB,BassikMC.DirectedevolutionusingdCas9-targetedsomatichypermutationinmammaliancells

RecruitingahyperactivecytidinedeaminaseviatheguideRNAtodCas9allowsfortheintroductionofdiversepointmutationsattheCRISPRtargetlocustocreatecomplexlibrariesofvariantsforproteinengineeringordissectionofproteinfunction.

KuscuC,AdliM.CRISPR-Cas9-AIDbaseeditorisapowerfulgain-of-functionscreeningtool

AbstractCombiningCRISPR-Cas9withanenzymethatinducessomatichypermutationsallowstherapidgenerationofdiversevariantsforgain-of-functionscreens.

QiLS,LarsonMH,GilbertLA,DoudnaJA,WeissmanJS,ArkinAP,LimWA.RepurposingCRISPRasanRNA-guidedplatformforsequence-specificcontrolofgeneexpression

GilbertLA,LarsonMH,MorsutL,LiuZ,BrarGA,TorresSE,Stern-GinossarN,BrandmanO,WhiteheadEH,DoudnaJA,LimWA,WeissmanJS,QiLS.CRISPR-mediatedmodularRNA-guidedregulationoftranscriptionineukaryotes

ChengAW,WangH,YangH,ShiL,KatzY,TheunissenTW,RangarajanS,ShivalilaCS,DadonDB,JaenischR.MultiplexedactivationofendogenousgenesbyCRISPR-on,anRNA-guidedtranscriptionalactivatorsystem

AbudayyehOO,GootenbergJS,KonermannS,JoungJ,SlaymakerIM,CoxDB,ShmakovS,MakarovaKS,SemenovaE,MinakhinL,SeverinovK,RegevA,LanderES,KooninEV,ZhangF.C2c2isasingle-componentprogrammableRNA-guidedRNA-targetingCRISPReffector

TheCRISPR-CasadaptiveimmunesystemdefendsmicrobesagainstforeigngeneticelementsviaDNAorRNA-DNAinterference.WecharacterizetheClass2typeVI-ACRISPR-CaseffectorC2c2anddemonstrateitsRNA-guidedRNasefunction.C2c2fromthebacteriumLeptotrichiashahiiprovidesinterferenceagainstRNAphage.InvitrobiochemicalanalysisshowthatC2c2isguidedbyasinglecrRNAandcanbeprogrammedtocleavessRNAtargetscarryingcomplementaryprotospacers.Inbacteria,C2c2canbeprogrammedtoknockdownspecificmRNAs.CleavageismediatedbycatalyticresiduesinthetwoconservedHEPNdomains,mutationsinwhichgeneratecatalyticallyinactiveRNA-bindingproteins.TheseresultsbroadenourunderstandingofCRISPR-CassystemsandsuggestthatC2c2canbeusedtodevelopnewRNA-targetingtools.

SmargonAA,CoxDBT,PyzochaNK,ZhengK,SlaymakerIM,GootenbergJS,AbudayyehOA,EssletzbichlerP,ShmakovS,MakarovaKS,KooninEV,ZhangF.Cas13bisatypeVI-BCRISPR-associatedRNA-GuidedRNasedifferentiallyregulatedbyaccessoryoroteinsCsx27andCsx28

ShmakovS,SmargonA,ScottD,CoxD,PyzochaN,YanW,AbudayyehOO,GootenbergJS,MakarovaKS,WolfYI,SeverinovK,ZhangF,KooninEV.Diversityandevolutionofclass2CRISPR-Cassystems

Class2CRISPR-Cassystemsarecharacterizedbyeffectormodulesthatconsistofasinglemultidomainprotein,suchasCas9orCpf1.Wedesignedacomputationalpipelineforthediscoveryofnovelclass2variantsandusedittoidentifysixnewCRISPR-Cassubtypes.Thediversepropertiesofthesenewsystemsprovidepotentialforthedevelopmentofversatiletoolsforgenomeeditingandregulation.InthisAnalysisarticle,wepresentacomprehensivecensusofclass2typesandclass2subtypesincompleteanddraftbacterialandarchaealgenomes,outlineevolutionaryscenariosfortheindependentoriginofdifferentclass2CRISPR-Cassystemsfrommobilegeneticelements,andproposeanamendedclassificationandnomenclatureofCRISPR-Cas.

YanWX,ChongS,ZhangH,MakarovaKS,KooninEV,ChengDR,ScottDA.Cas13disacompactRNA-targetingtypeVICRISPReffectorpositivelymodulatedbyaWYL-domain-containingaccessoryprotein

GootenbergJS,AbudayyehOO,KellnerMJ,JoungJ,CollinsJJ,ZhangF.MultiplexedandportablenucleicaciddetectionplatformwithCas13,Cas12a,andCsm6

AbstractRapiddetectionofnucleicacidsisintegralforclinicaldiagnosticsandbiotechnologicalapplications.WerecentlydevelopedaplatformtermedSHERLOCK(SpecificHighSensitivityEnzymaticReporterUnLOCKing)thatcombinesisothermalpre-amplificationwithCas13todetectsinglemoleculesofRNAorDNA.ThroughcharacterizationofCRISPRenzymologyandapplicationdevelopment,wereportherefouradvancesintegratedintoSHERLOCKv2:1)4-channelsinglereactionmultiplexingusingorthogonalCRISPRenzymes;2)quantitativemeasurementofinputdownto2aM;3)3.5-foldincreaseinsignalsensitivitybycombiningCas13withCsm6,anauxilaryCRISPR-associatedenzyme;and4)lateralflowread-out.SHERLOCKv2candetectDengueorZikavirusssRNAaswellasmutationsinpatientliquidbiopsysamplesvialateralflow,highlightingitspotentialasamultiplexable,portable,rapid,andquantitativedetectionplatformofnucleicacids.

ShalemO,SanjanaNE,ZhangF.High-throughputfunctionalgenomicsusingCRISPR-Cas9

LiuSJ,HorlbeckMA,ChoSW,BirkHS,MalatestaM,HeD,AttenelloFJ,VillaltaJE,ChoMY,ChenY,MandegarMA,OlveraMP,GilbertLA,ConklinBR,ChangHY,WeissmanJS,LimDA.CRISPRi-basedgenome-scaleidentificationoffunctionallongnoncodingRNAlociinhumancells

ThehumangenomeproducesthousandsoflongnoncodingRNAs(lncRNAs)-transcripts>200nucleotideslongthatdonotencodeproteins.AlthoughcriticalrolesinnormalbiologyanddiseasehavebeenrevealedforasubsetoflncRNAs,thefunctionofthevastmajorityremainsuntested.WedevelopedaCRISPRinterference(CRISPRi)platformtargeting16,401lncRNAlociinsevendiversecelllines,includingsixtransformedcelllinesandhumaninducedpluripotentstemcells(iPSCs).Large-scalescreeningidentified499lncRNAlocirequiredforrobustcellulargrowth,ofwhich89%showedgrowth-modifyingfunctionexclusivelyinonecelltype.WefurtherfoundthatlncRNAknockdowncanperturbcomplextranscriptionalnetworksinacelltype-specificmanner.ThesedataunderscorethefunctionalimportanceandcelltypespecificityofmanylncRNAs.

HanK,JengEE,HessGT,MorgensDW,LiA,BassikMC.SynergisticdrugcombinationsforcanceridentifiedinaCRISPRscreenforpairwisegeneticinteractions

NajmFJ,StrandC,DonovanKF,HegdeM,SansonKR,VaimbergEW,SullenderME,HartenianE,KalaniZ,FusiN,ListgartenJ,YoungerST,BernsteinBE,RootDE,DoenchJG.OrthologousCRISPR-Cas9enzymesforcombinatorialgeneticscreens

AbstractCombinatorialgeneticscreeningusingCRISPR-Cas9isausefulapproachtouncoverredundantgenesandtoexplorecomplexgenenetworks.However,currentmethodssufferfrominterferencebetweenthesingle-guideRNAs(sgRNAs)andfromlimitedgenetargetingactivity.Toincreasetheefficiencyofcombinatorialscreening,weemployorthogonalCas9enzymesfromStaphylococcusaureusandStreptococcuspyogenes.WeusedmachinelearningtoestablishS.aureusCas9sgRNAdesignrulesandpairedS.aureusCas9withS.pyogenesCas9toachievedualtargetinginahighfractionofcells.Wealsodevelopedalentiviralvectorandcloningstrategytogeneratehigh-complexitypooleddual-knockoutlibrariestoidentifysyntheticlethalandbufferinggenepairsacrossmultiplecelltypes,includingMAPKpathwaygenesandapoptoticgenes.Ourorthologousapproachalsoenabledascreencombininggeneknockoutswithtranscriptionalactivation,whichrevealedgeneticinteractionswithTP53.The"BigPapi"(pairedaureusandpyogenesforinteractions)approachdescribedherewillbewidelyapplicableforthestudyofcombinatorialphenotypes.

JoungJ,EngreitzJM,KonermannS,AbudayyehOO,VerdineVK,AguetF,GootenbergJS,SanjanaNE,WrightJB,FulcoCP,TsengYY,YoonCH,BoehmJS,LanderES,ZhangF.Genome-scaleactivationscreenidentifiesalncRNAlocusregulatingageneneighbourhood

AbstractThiscorrectsthearticleDOI:10.1038/nature23451.

SanjanaNE,ShalemO,ZhangF.Improvedvectorsandgenome-widelibrariesforCRISPRscreening

XueW,ChenS,YinH,TammelaT,PapagiannakopoulosT,JoshiNS,CaiW,YangG,BronsonR,CrowleyDG,ZhangF,AndersonDG,SharpPA,JacksT.CRISPR-mediateddirectmutationofcancergenesinthemouseliver

ChoiPS,MeyersonM.TargetedgenomicrearrangementsusingCRISPR/Castechnology

Genomicrearrangementsarefrequentlyobservedincancercellsbuthavebeendifficulttogenerateinahighlyspecificmannerforfunctionalanalysis.HerewereporttheapplicationofCRISPR/Castechnologytosuccessfullygenerateseveraltypesofchromosomalrearrangementsimplicatedasdrivereventsinlungcancer,includingtheCD74-ROS1translocationeventandtheEML4-ALKandKIF5B-RETinversionevents.OurresultsdemonstratethatCas9-inducedDNAbreakspromoteefficientrearrangementbetweenpairsoftargetedloci,providingahighlytractableapproachforthestudyofgenomicrearrangements.

ZuckermannM,HovestadtV,Knobbe-ThomsenCB,ZapatkaM,NorthcottPA,SchrammK,BelicJ,JonesDT,TschidaB,MoriarityB,LargaespadaD,RousselMF,KorshunovA,ReifenbergerG,PfisterSM,LichterP,KawauchiD,GronychJ.SomaticCRISPR/Cas9-mediatedtumoursuppressordisruptionenablesversatilebraintumourmodelling

CarrollKJ,MakarewichCA,McanallyJ,AndersonDM,ZentilinL,LiuN,GiaccaM,Bassel-DubyR,OlsonEN.Amousemodelforadultcardiac-specificgenedeletionwithCRISPR/Cas9

PaquetD,KwartD,ChenA,SproulA,JacobS,TeoS,OlsenKM,GreggA,NoggleS,Tessier-LavigneM.EfficientintroductionofspecifichomozygousandheterozygousmutationsusingCRISPR/Cas9

GootenbergJS,AbudayyehOO,LeeJW,EssletzbichlerP,DyAJ,JoungJ,VerdineV,DonghiaN,DaringerNM,FreijeCA,MyhrvoldC,BhattacharyyaRP,LivnyJ,RegevA,KooninEV,HungDT,SabetiPC,CollinsJJ,ZhangF.NucleicaciddetectionwithCRISPR-Cas13a/C2c2

WangHX,LiM,LeeCM,ChakrabortyS,KimHW,BaoG,LeongKW.CRISPR/Cas9-basedgenomeeditingfordiseasemodelingandtherapy:Challengesandopportunitiesfornonviraldelivery

DunbarCE,HighKA,JoungJK,KohnDB,OzawaK,SadelainM.Genetherapycomesofage

Advancesintheunderstandingofmolecularbiologyofhumandiseaseandthedevelopmentofefficientgenetransfertechniqueshaveresultedinpracticalapproachestohumangenetherapy,withnewtechniquesbeingdevelopedatanincreasingrate.Thefirsttrialshavenowbeguninhumansandinitialresultsarepositive.

BouladF,Mansilla-SotoJ,CabrioluA,RiviereI,SadelainM.GeneTherapyandgenomeediting

Chimericantigenreceptors(CARs)aresyntheticreceptorsthatreprogramTlymphocytestotargetchosenantigens.ThetargetingofCD19,acellsurfacemoleculeexpressedinthevastmajorityofleukemiasandlymphomas,hasbeensuccessfullytranslatedintheclinic,earningCARtherapyaspecialdistinctionintheselectionof"cancerimmunotherapy"byScienceasthebreakthroughoftheyear...[Showfullabstract]

Mansilla-SotoJ,RiviereI,BouladF,SadelainM.CellandGeneTherapyforthebeta-thalassemias:advancesandprospects

YinH,XueW,ChenS,BogoradRL,BenedettiE,GrompeM,KotelianskyV,SharpPA,JacksT,AndersonDG.GenomeeditingwithCas9inadultmicecorrectsadiseasemutationandphenotype

AbstractWedemonstrateCRISPR-Cas9-mediatedcorrectionofaFahmutationinhepatocytesinamousemodelofthehumandiseasehereditarytyrosinemia.DeliveryofcomponentsoftheCRISPR-Cas9systembyhydrodynamicinjectionresultedininitialexpressionofthewild-typeFahproteinin0908041/250livercells.ExpansionofFah-positivehepatocytesrescuedthebodyweightlossphenotype.OurstudyindicatesthatCRISPR-Cas9-mediatedgenomeeditingispossibleinadultanimalsandhaspotentialforcorrectionofhumangeneticdiseases.

DeverDP,BakRO,ReinischA,CamarenaJ,WashingtonG,NicolasCE,Pavel-DinuM,SaxenaN,WilkensAB,MantriS,UchidaN,HendelA,NarlaA,MajetiR,WeinbergKI,PorteusMH.CRISPR/Cas9beta-globingenetargetinginhumanhaematopoieticstemcells

TabebordbarM,ZhuK,ChengJKW,ChewWL,WidrickJJ,YanWX,MaesnerC,WuEY,XiaoR,RanFA,CongL,ZhangF,VandenbergheLH,ChurchGM,WagersAJ.Invivogeneeditingindystrophicmousemuscleandmusclestemcells

NelsonCE,HakimCH,OusteroutDG,ThakorePI,MorebEA,CastellanosRiveraRM,MadhavanS,PanX,RanFA,YanWX,AsokanA,ZhangF,DuanD,GersbachCA.InvivogenomeeditingimprovesmusclefunctioninamousemodelofDuchennemusculardystrophy

Duchennemusculardystrophy(DMD)isadevastatingdiseaseaffectingabout1outof5000malebirthsandcausedbymutationsinthedystrophingene.Genomeeditinghasthepotentialtorestoreexpressionofamodifieddystrophingenefromthenativelocustomodulatediseaseprogression.Inthisstudy,adeno-associatedviruswasusedtodelivertheclusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)-Cas9systemtothemdxmousemodelofDMDtoremovethemutatedexon23fromthedystrophingene.Thisincludeslocalandsystemicdeliverytoadultmiceandsystemicdeliverytoneonatalmice.Exon23deletionbyCRISPR-Cas9resultedinexpressionofthemodifieddystrophingene,partialrecoveryoffunctionaldystrophinproteininskeletalmyofibersandcardiacmuscle,improvementofmusclebiochemistry,andsignificantenhancementofmuscleforce.ThisworkestablishesCRISPR-Cas9-basedgenomeeditingasapotentialtherapytotreatDMD.

LongC,AmoasiiL,MireaultAA,McanallyJR,LiH,Sanchez-OrtizE,BhattacharyyaS,SheltonJM,Bassel-DubyR,OlsonEN.Postnatalgenomeeditingpartiallyrestoresdystrophinexpressioninamousemodelofmusculardystrophy

GaoX,TaoY,LamasV,HuangM,YehWH,PanB,HuYJ,HuJH,ThompsonDB,ShuY,LiY,WangH,YangS,XuQ,PolleyDB,LibermanMC,KongWJ,HoltJR,ChenZY,LiuDR.Treatmentofautosomaldominanthearinglossbyinvivodeliveryofgenomeeditingagents

HawksworthJ,SatchwellTJ,MeindersM,DanielsDE,ReganF,ThorntonNM,WilsonMC,DobbeJG,StreekstraGJ,TrakarnsangaK,HeesomKJ,AnsteeDJ,FrayneJ,ToyeAM.EnhancementofredbloodcelltransfusioncompatibilityusingCRISPR-mediatederythroblastgeneediting

HuangYQ,LiGL,YangHQ,WuZF.Progressandapplicationofgenome-editedpigsinbiomedicalresearch

黄耀强,李国玲,杨化强,吴珍芳.基因编辑猪在生物医学研究中的应用

ZhangDW,ZhangCF,DongF,HuangYL,ZhangY,ZhouH.ApplicationofCRISPR/Cas9systeminbreedingofnewantiviralplantgermplasm

张道微,张超凡,董芳,黄艳岚,张亚,周虹.CRISPR/Cas9系统在培育抗病毒植物新种质中的应用

LiJ,MengX,ZongY,ChenK,ZhangH,LiuJ,LiJ,GaoC.GenereplacementsandinsertionsinricebyintrontargetingusingCRISPR-Cas9

ShiJ,GaoH,WangH,LafitteHR,ArchibaldRL,YangM,HakimiSM,MoH,HabbenJE.ARGOS8variantsgeneratedbyCRISPR-Cas9improvemaizegrainyieldunderfielddroughtstressconditions

ZhouH,HeM,LiJ,ChenL,HuangZ,ZhengS,ZhuL,NiE,JiangD,ZhaoB,ZhuangC.Developmentofcommercialthermo-sensitivegenicmalesterilericeaccelerateshybridricebreedingusingtheCRISPR/Cas9-mediatedTMS5editingsystem

SunY,ZhangX,WuC,HeY,MaY,HouH,GuoX,DuW,ZhaoY,XiaL.Engineeringherbicide-resistantriceplantsthroughCRISPR/Cas9-mediatedhomologousrecombinationofacetolactatesynthase

WangM,LuY,BotellaJR,MaoY,HuaK,ZhuJK.GeneTargetingbyhomology-directedrepairinriceusingageminivirus-basedCRISPR/Cas9system

HaiT,TengF,GuoR,LiW,ZhouQ.One-stepgenerationofknockoutpigsbyzygoteinjectionofCRISPR/Cassystem

One-stepgenerationofknockoutpigsbyzygoteinjectionofCRISPR/CassystemCellResearchadvanceonlinepublication,January312014.doi:10.1038/cr.2014.11Authors:TangHai,Fei...

LiuX,WangY,TianY,YuY,GaoM,HuG,SuF,PanS,LuoY,GuoZ,QuanF,ZhangY.Generationofmastitisresistanceincowsbytargetinghumanlysozymegenetobeta-caseinlocususingzinc-fingernucleases

LuoJ,SongZ,YuS,CuiD,WangB,DingF,LiS,DaiY,LiN.Efficientgenerationofmyostatin(MSTN)biallelicmutationsincattleusingzincfingernucleases

NiW,QiaoJ,HuS,ZhaoX,RegouskiM,YangM,PolejaevaIA,ChenC.EfficientgeneknockoutingoatsusingCRISPR/Cas9system

AbstractTheCRISPR/Cas9systemhasbeenadaptedasanefficientgenomeeditingtoolinlaboratoryanimalssuchasmice,rats,zebrafishandpigs.Here,wereportthatCRISPR/Cas9mediatedapproachcanefficientlyinducemonoallelicandbiallelicgeneknockoutingoatprimaryfibroblasts.FourgenesweredisruptedsimultaneouslyingoatfibroblastsbyCRISPR/Cas9-mediatedgenomeediting.Thesingle-geneknockoutfibroblastsweresuccessfullyusedforsomaticcellnucleartransfer(SCNT)andresultedinlive-borngoatsharboringbiallelicmutations.TheCRISPR/Cas9systemrepresentsahighlyeffectiveandfacileplatformfortargetededitingoflargeanimalgenomes,whichcanbebroadlyappliedtobothbiomedicalandagriculturalapplications.

CuiC,SongY,LiuJ,GeH,LiQ,HuangH,HuL,ZhuH,JinY,ZhangY.GenetargetingbyTALEN-inducedhomologousrecombinationingoatsdirectsproductionofbeta-lactoglobulin-free,high-humanlactoferrinmilk

QianL,TangM,YangJ,WangQ,CaiC,JiangS,LiH,JiangK,GaoP,MaD,ChenY,AnX,LiK,CuiW.Targetedmutationsinmyostatinbyzinc-fingernucleasesresultindouble-muscledphenotypeinMeishanpigs

WangK,OuyangH,XieZ,YaoC,GuoN,LiM,JiaoH,PangD.EfficientgenerationofmyostatinmutationsinpigsusingtheCRISPR/Cas9system

WhitworthKM,RowlandRR,EwenCL,TribleBR,KerriganMA,Cino-OzunaAG,SamuelMS,LightnerJE,MclarenDG,MilehamAJ,WellsKD,PratherRS.Gene-editedpigsareprotectedfromporcinereproductiveandrespiratorysyndromevirus

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1.科研网站汇总转载科研网站汇总-转载 1、DEEPL翻译:https://www.deepl.com/translator 2、Word hippo:https://www.wordhippo.com/ 3、检查语法:https://virtualwritingtutor.com/ 4、X-mol:https://www.x-mol.com/ 5、文献部落:https://www.x-mol.com/ 6、大木虫:http://www.4243.net/https://zhuanlan.zhihu.com/p/11750038860
2.HomeMicrosoft Research Find My Things: New teachable AI tool helps blind and low-vision people locate lost personal items View our story Featured Blogs Dec 4 Research Focus: Week of December 2, 2024 Publication Featured Blogs Dec 4 Downloadhttp://research.microsoft.com/en-us/um/people/jrzhou/
3.CRISPRTarget:bioinformaticpredictionandanalysisofcrRNAThe bacterial and archaeal CRISPR/Cas adaptive immune system targets specific protospacer nucleotide sequences in invading organisms. This requires base pairing between processed CRISPR RNA and the target protospacer. For type I and II CRISPR/Cas systemshttps://www.ncbi.nlm.nih.gov/pubmed/23492433
4.ScalablecharacterizationofthePAMrequirementsofCRISPRThe continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR–Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assayhttps://www.nature.com/articles/s41596-020-00465-2
5.大肠埃希菌CRISPRs间隔序列的来源研究【摘要】:目的研究大肠埃希菌全基因组中规律成簇的间隔短回文重复序列(CRISPRs)的间隔序列分布及来源规律。方法通过BLAST、CRISPRs finder、ClustalX和CRISPRTarget分析CRISPR/Cas、间隔序列及其来源。结果 203株大肠埃希菌中74.4%含有I-E CRISPR/Cas,9.4%含有I-F CRISPR/Cas,17.2%存在CRISPR3-4;CRISPR1,CRISPR2,https://www.cnki.com.cn/Article/CJFDTotal-ZISC201902003.htm
6.genomeeditingbasedonthesubtypeIBSviCRISPRNotably, no off-target changes or indel-formation were detected in the analysis of potential off-target sites. This discovery broadens our understanding of the diversity of type I CRISPR-Cas systems and will facilitate new developments in genome editing tools. Keywords Self- and non-self-targetinghttp://arxiv.org/pdf/2305.05093
7.CRISPR/CasHere, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition, followed by extracellular transduction and https://pubs.acs.org/doi/10.1021/acsami.3c07918
8.CRISPR(protein assistant motif) is a number of nucleotides adjacent to the target site, which is very important for the Cas protein to recognize the target sequence and is also the key characteristic of CRISPR-Cas. There are several reported methods for identification of PAM. In this review, we https://www.360doc.cn/article/3843034_1043712356.html
9.刘佳课题组构建针对膜蛋白sgRNA设计的CRISPR网站CRISPR近日,上海科技大学免疫化学研究所、生命科学与技术学院刘佳课题组与免疫化学研究所生物医学大数据平台合作,通过升级sgRNA设计的算法,针对已被质谱鉴定的细胞表面蛋白基因设计sgRNA得到膜蛋白组sgRNA文库(CRISPR-Surfaceome),创建了网上数据库CRISPR-Surfaceome(https://crispr-surfaceome.siais.shanghaitech.edu.cn/home)。https://siais.shanghaitech.edu.cn/2022/0815/c5404a767972/page.htm
10.CRISPR网站设计gRNA数据怎样分析呢分子生物如果一列预测的efficiency 是60 一列是10 你需要注意了,这种设计的gRNA很有可能不会作用,off target https://muchong.com/t-13645661-1
11.sgRNAs&基因编辑sgrna设计网站Off-Target也就是脱靶效应,由于CRISPR技术的切割是由sgRNA根据PAM序列定位识别位点,从而进行切割,但是如果sgRNA识别的位点是错误的,就产生了错误切割,这样就产生了脱靶效应。所以说,在进行sgRNA选择的时候,要尽可能地选择脱靶效率比较低的,也就是Off-target分值比较高的。另外,符合sgRNA的其它设计原则就可以了。https://blog.csdn.net/geekfocus/article/details/128613082
12.CRISPR/Spacer/Cas有关预测方法:集锦细菌利用记录在CRISPR间的病毒Spacers,快速识别入侵病毒,激活Cas蛋白切割病毒DNA保护自己。收罗了一些有关CRISPR/Spacer/Cas预测的方法。 CRISPR-Cas9系统简介 方法期刊时间被引 CRISPRFinder Nucleic Acids Res. 2007 1732 CRISPRCasFinder Nucleic Acids Res. 2018 482 CRISPRTarget RNA Biology 2013 231 MacSyFinder https://www.jianshu.com/p/b17ff3d1bfe7
13.CRRSPR/Cas9基因敲除系统之sgRNA设计sgRNA设计网站介绍 sgRNA的在线设计网站有: 1、http://crispr.mit.edu/; 2、http://www.e-crisp.org/E-CRISP/; 等网站。这些网站虽然一定程度上满足了部分研究 者的需要,但仍然存在很多不足和缺陷。例如: 1、数据分析周期长; 2、无具体的脱靶数据; https://www.biomart.cn/experiment/443/680/206473.htm
14.植物基因组编辑新工具——引导编辑技术文中介绍了植物引导编辑的开发历程、组成结构、优点和局限性,并重点介绍了植物引导编辑效率优化的进展,包括Tm值指导的PBS序列设计、RT模板长度、双pegRNA策略、PlantPegDesigner网站的开发和PPE效应蛋白优化策略。最后,对植物引导编辑技术的未来发展和应用提出了展望。https://cjb.ijournals.cn/html/cjbcn/2022/1/gc22010026.htm
15.GSK组织GeneDisco挑战赛,探索体外基因实验的广阔设计空间GSK在其官方网站上发布了GeneDisco挑战赛,2022年3月31日提交截止。以下是简要内容。 介绍 利用CRISPR技术等进行基因干预的体外细胞实验,是早期药物发现和靶点验证的一个重要步骤,有助于评估关于生物机制和疾病病理之间因果关系的初步假设。由于有数十亿个潜在的假设需要测试,体外遗传实验的实验设计空间极其巨大,而现有的https://cloud.tencent.com/developer/article/1980296
16.可视化CRISPR/Cas9基因编辑系统及使用方法81.一种实施例1的可视化剔除转基因成分的crispr/cas9基因编辑的使用方法,应用的具体对象为水稻穗颈伸长基因oseui1,具体包括以下步骤: 82.(1)利用http://skl.scau.edu.cn/targetdesign/网站targetdesign工具设计oseui1基因的2个靶位点eui1 ? u3 ? https://www.xjishu.com/zhuanli/27/202110679391.html
17.世界首个完全由AI设计的CRISPR基因编辑器来了!医药新闻2024年4月23日,寻百会生物(GV20 Therapeutics)在Cell上发表了题为:IGSF8 is an innate immune checkpoint and cancer immunotherapy target的研究论文,报道了去年,首款基于CRISPR的基因编辑疗法获批上市,标志着遗传疾病治疗的新纪元。与此同时,人工智能的进步也为设计更强大的基因编辑器带来了希望。https://bydrug.pharmcube.com/news/detail/caaf4f23c9805c777a3c7947e7f137f4
18.介绍有用的CRISPRsgRNA设计软件细胞生物与生物信息你们得到KO line的速度挺快的,有没什么好的经验可以分享一下呢? 比如 药物筛选时间,3天以上? Cas9和sgRNA转染比例? sgRNAcas9程序可以设计 Double Nick (程序中设置为 paired-sgRNA searching model),有兴趣可以试用一下。Source from CELL paper:Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced https://3g.dxy.cn/bbs/topic/30624273
19.CRISPR/Cas9载体设计与构建的作用机理CRISPR/Cas9 载体设计与构建方法2:根据NtDXR基因序列,利用在线工具ZiFiT Targeter Version4.2:选择合适的靶位点,筛选要求为:①靶位点主要包括20 个碱基,并且这20 个碱基后面是NGG(N 为任意碱基)3 个碱基的PAM 区(Protospacer adjacent motif,PAM);②靶位点尽量选择在基因编码区的前端。依据靶位点设计原则设计引物,https://mip.chemicalbook.com/NewsInfo_5909.htm
20.CRISPR选择“CRISPR/Cas9”模式,设置完 /targetFinder/) 。有2 种设计模 参数后,点击“寻找靶标结合位点( Find Target Sites!)” 式,其一是可针对序列设计sgRNA 并评估脱靶效应, 按钮,软件即可运行。结果输出会将sgRNA 标示到 其次是预测已知 sgRNA 的脱靶效应。允许输入的 目标基因组的染色体上,并以不同颜色标示其https://max.book118.com/html/2017/0920/134497738.shtm
21.GenerationofNonhumanPrimateModelofConeDysfunctionThe regions surrounding the CRISPR target site were amplified with Phanta Max Super-Fidelity DNA Polymerase. The PCR product was denatured at 95°C for 5 min and gradually reannealed to form DNA heteroduplexes. The heteroduplexes were digested with T7 endonuclease I (NEB, Shanghai, China) https://www.sciencedirect.com/science/article/pii/S232905012030173X
22.Nature细胞命运重塑的新篇章:染色质结构与重编程因子的协奏细胞命运的转换是现代生命科学和医学领域中最具革命性的话题之一。通过精准操控细胞命运,研究人员可以将一种成熟的细胞类型重编程为另一种完全不同的细胞类型,这种过程不仅揭示了生命系统的可塑性,更为再生医学、疾病建模和个性化治疗开辟了新https://mp.weixin.qq.com/s?__biz=MzU2MTQ2MDE0Ng==&mid=2247579274&idx=2&sn=32040630ee45c3ee77a8cd6c87b06f91&chksm=fd138021e94eb5f137762aaad34d04982f58aaa5bbe83c430f9b32be621fe15b34c3813b3c50&scene=27