基因打靶和遺傳修飾動物模型課件

基因打靶和遺傳修飾動物模型TheGeneTargetingandHeredity-ModifiedAnimal

ModelIntroductionGeneTargeting(Knock

out)HomologousrecombinationZincFingerNuclease,

ZFNTranscription-Activator-LikeEffectorNuclease,TALENClusteredRegulatoryInterspacedShortPalindromicRepeats(CRISPR)sequenceswithCRISPR-AssociatedProtein9(Cas9),

CRISPR/Cas9TheNgAgo–gDNA

systemStructureguideendonuclease(SNG)

systemIntroductionTransgenetictechnology(Knock

in)random

integrationsIntroductionGenetargetinginmouseembryonicstemcellsisnowawell-establishedtechniquethatiswidelyusedtocreateanimalmodelsfor

humandiseaseortostudygenefunctionatthelevelofthewhole

animal.概

述基因敲除是自80年代末以來發(fā)展起來的一種新型分子生物學(xué)技術(shù)，是通過一定的途徑使機(jī)體特定的基因失活或缺失的技術(shù)。通常意義上的基因敲除主要是應(yīng)用DNA同源重組原理，用設(shè)計的同源片段替代靶基因片段，從而達(dá)到基因敲除的目的。概

述隨著基因敲除技術(shù)的發(fā)展，除了同源重組外，新的原理和技術(shù)也逐漸被應(yīng)用，比較成功的有ZFN、TALEN、CRISPR/Cas9、The

NgAgo–gDNAsystem

，它們同樣可以達(dá)到基因敲除的目的。TheBIG

BreakthroughsDiscoverythatEScellsderivedfrompre-implantationembryos(blastocysts)werepluripotent(MartinEvansin

1981).Discoverythatmammaliancellsinculturecould

undergohomologousrecombinationwithgeneticallyengineeredDNA(MarioCapecchiandOliverSmithiesinthe

1980’s).TheBIG

Breakthroughs到1987年，Thompsson首次建立了完整的ES細(xì)胞基因敲除的小鼠模型。到目前為止，運用基因同源重組、非同源末端連接（non-homologous

end-joining,NHEJ）或同源指導(dǎo)修復(fù)（homology-directed

repair,

HDR）進(jìn)行基因敲除（替換）依然是構(gòu)建基因敲除（遺傳修飾）動物模型中最普遍的使用方法。實現(xiàn)基因敲除的多種原理和方法利用基因同源重組進(jìn)行基因敲除利用基因同源重組進(jìn)行基因敲除條件性基因敲除法條件性基因敲除法誘導(dǎo)性基因敲除法誘導(dǎo)性基因敲除法利用隨機(jī)插入突變進(jìn)行基因敲除利用隨機(jī)插入突變進(jìn)行基因敲除基因捕獲法基因捕獲法RNA

interference，RNAi引起的基因敲除。ZFN,TALEN,CRISPR/Cas9,NgAgo–gDNAsystem,SNG

systemRNA

interference，RNAi引起的基因敲除。ZFN,TALEN,CRISPR/Cas9,NgAgo–gDNAsystem,SNG

system同源重組同源重組（Homologus

Recombination）

是指發(fā)生在非姐妹染色單體（sister

chromatin）

之間或同一染色體上含有同源序列的DNA分子之間或分子之內(nèi)的重新組合。同源重組需要一系列的蛋白質(zhì)催化，如原核生物細(xì)胞內(nèi)的RecA、RecBCD、RecF、RecO、RecR等；以及真核生物細(xì)胞內(nèi)的Rad51、Mre11-Rad50等等。Homologus

RecombinationHomologousrecombinationwithtransfecteddisruptionconstructscaninactivatespecifictargetgenesinES

cells.Whenrecipientdiploidcellsaretransformedwiththegenedisruptionconstruct,homologousrecombinationbetweentheendsoftheconstructandthecorrespondingchromosomalsequenceswillintegrategeneintothechromosome,replacingthetargetgenesequence.Homologousrecombinationwithtransfecteddisruptionconstructscaninactivatespecifictargetgenesin

cells.利用同源重組構(gòu)建基因敲除動物模型的基本步驟得到純合體基因載體的構(gòu)建ES

細(xì)胞的獲得同源重組選擇篩選已擊中的細(xì)胞表型研究得到純合體1.Vector

constructionConstructfordisruptingatargetgenecanbepreparedbythe

PCR.Thetwoprimersdesignedforthispurposeeachcontainasequenceofabout20nucleotides(nt)thatishomologoustooneendofthetargetyeastgeneaswellassequencesneededtoamplifyasegmentofDNAcarryingaselectablemarkergenesuchasNeomycinR,whichconfersresistanceto

G-418.Neomycin

RNeomycin

R1.Vector

constructionNeomycinbelongstoaminoglycosideclassofantibioticsthatcontaintwoormoreaminosugarsconnectedbyglycosidic

bonds.1.Vector

constructionG418(Geneticin)isanaminoglycosideantibiotic.G418blockspolypeptidesynthesisbyinhibitingtheelongationstepinbothprokaryoticandeukaryotic

cells.1.Vector

constructionResistancetoG418isconferredbythe

neogene

fromTn5encodinganaminoglycoside3'-phosphotransferase,APT3'II.Thebacterialneomycinphosphotransferase(neo)gene.Isolatedfromapre-implantation

embryosFrominnercellmassofblastulastage

embryoCellsare

undifferentiated

Cellsare

pluripotent

Abletodifferentiateintomany

(all?)differentcelltypesin

embryo.2.EmbryonicStemCell

IsolationIsolatedfromapre-implantation

embryosgrownincultureneedcellstodividebutnotdifferentiatelongertimeinculture=moredifferentiation.2.EmbryonicStemCell

Isolation2.EmbryonicStemCell

IsolationInnerCell

Mass3.Homologous

recombinationGeneTargeting

Technology

Cellstakeupexogenous

DNAFrequencyisvery

lowCanbeinducedtohigherfrequency(chemical,electrical,

injection)

InDividing

Cells:SomeDNAincorporatedinto

genomeRandomintegration—rare

eventHomologousRecombination—evenmore

rare3.Homologous

recombinationRandomIntegration:

DNAincorporatedanywhereingenome (nottargeted)

copynumbercanbevery

high.

disruptstheendogenousDNAatinsertion

site.

transgenicanimalsareusuallyrandom

integrations.3.Homologous

recombinationdrivenbytheDNAsequences

(targeted)rare(1000×

lessfrequentthan

random)increasedefficiency:isogenicDNAlongerstretchesof

homologyresults

in:endogenousDNAreplacedwith

exogenousspecific-targetedcopynumber=

13.Homologous

recombination3.Homologous

recombinationabcda'b'c'd'abc'd'3.Homologous

recombinationNon-homologousDNAFlankedbyHomologous

DNAabcda'b'c'd'ab'c'd'3.Homologous

recombinationCre/loxPSystemorCre-Lox

recombinationCre(CausesRecombination);loxP(locusofcrossing(x)over,

P1)

CreRecombinaseisatyrosinerecombinaseenzymederivedfromtheP1Bacteriophage.TheenzymeusesatopoisomeraseIlikemechanismtocarryoutsitespecificrecombination

events.3.Homologous

recombinationCre/loxPSystemorCre-Lox

recombinationCre(CausesRecombination);loxP(locusofcrossing(x)over,

P1)

Theenzyme(38kDa)isamemberoftheIntegrasefamilyofsitespecificrecombinaseanditisknowntocatalysethesitespecificrecombinationeventbetweentwoDNArecognitionsites(loxPsites).NagyA(2000)."CreRecombinase:TheUniversalReagentforGenomeTailoring".Genesis26:

99–109.3.Homologous

recombinationCre/loxPSystemCre-Lox

recombinationCre(CausesRecombination);loxP(locusofcrossing(x)over,

P1)ModelofCreRecombinaseboundtoitssubstrate

(DNA).Theaminoterminaldomainisshowninbluewhilstthecarboxyldomainisgreen3.Homologous

recombinationCre/loxPSystemorCre-Lox

recombinationloxP(locusofcrossing(x)over,

P1)LoxP(locusofX-overP1)isasiteontheBacteriophageP1consistingof34bp.Thereexistsanasymmetric8bpsequenceinbetweenwithtwosetsofpalindromic,13bpsequencesflankingit.Thedetailedstructureisgiven

below.13bp 8bp 13bpATAACTTCGTATA–GCATACAT–

TATACGAAGTTATTwoinverted13bprepeatssurrounding8bp

core.3.Homologous

recombinationCreMediated

RecombinationloxP loxP“floxed”CreandTwoseparateDNAspeciesbothcontainingloxPsitescanundergofusionastheresultofCremediatedrecombination.DNAsequencesfoundbetweentwoloxPsitesaresaidtobe

"floxed".3.Homologous

recombination“Floxing”aGeneinES

Cells23HSV-TKNeomycin

RNeomycin

R123loxP1231Vector3.Homologous

recombinationCreRecombinationinTargetedES

cellsNeomycin

R123TransientCre

expression13Neomycin

R1123OROR3usetomake

mouseloxP loxPloxP4.SelectionofrecombinantEScellsTargetingconstructforpositive/negativeselectionTomaketargetingconstruct:

Apositiveselectablemarkerflankedbytwo“arms”ofhomologous

sequence

Anegativeselectablemarkeroutsideonehomologous

arm4.SelectionofrecombinantEScellsTargetingconstructforpositive/negativeselection123413Neomycin

RHSV-TKVectorWhenexogenousDNAisintroducedintoembryonicstem(ES)cells,randominsertionvianonhomologousrecombinationoccursmuchmorefrequentlythangene-targetedinsertionviahomologousrecombination.RecombinantcellsinwhichonealleleofgeneX(orangeandwhite)isdisruptedcanbeobtainedbyusingarecombinantvectorthatcarriesgeneXdisruptedwithneor(green),whichconfersresistancetoG-418,and,outsidetheregionofhomology,tkHSV

(yellow).HerpesSimplexVirusThymidineKinase(HSV-TK)tkHSV(yellow),thethymidinekinasegenefromherpessimplex

virus.Theviralthymidinekinase,unliketheendogenousmouseenzyme,canconvertthenucleotideanalogganciclovirintothemonophosphateform;thisisthenmodifiedtothetriphosphateform,whichinhibitscellularDNAreplicationinES

cells.ThusgancicloviriscytotoxicforrandomrecombinantEScellscarryingthetkHSV

gene.GanciclovirorGancyclovir（更昔洛韋或丙氧鳥苷）4.SelectionofrecombinantEScellsSelection

Strategy:PositiveSelection–G418NeomycinResistancegeneconfersresistanceto

G418.

G418selectsfor

both:homologousandrandom

integrations.killscellsthathavenottakenup

DNA.NegativeSelection-

GancyclovirHerpesSimplexVirusThymidineKinase(HSV-TK)

.sensitivetogancyclovirselectsagainstrandomintegrants.PositiveandnegativeselectionofrecombinantEScellsPositiveandnegativeselectionofrecombinantEScellsCreMediated

Recombination123413Neomycin

RHSV-TK13 4Neomycin

R1 3 4Neomycin

RVectorRecombination,positive

andnegative

select.CreMediated

RecombinationTargeting:ElectroporateEScellswithTargetingConstruct.SelectinG418andGancyclovir—enrichesforhomologous

recombination.PickindividualcoloniesofresistantEScells(100s).Screenforproperlytargetedcells—PCRand/orSouthern

blot.Usetargetedcellstomakea

mouse.5.Insertthestemcellsintotheblastocystofa

mouse5.Insertthestemcellsintotheblastocystofa

mouse6.Phenotype

analysisWhyMakeaKnockout

Mouse?Determinethefunctionofaparticulargene/gene

product.?eliminateaspecific

protein.Examineaprotein’sroleinabiologicalprocess.?proteinXalwaysincreasedduringprocess

Y.WhyMakeaKnockout

Mouse?Determinefunctionofanewlyidentified

gene?genomesequencingprojects(≈25,000

genes)?manyofunknown

function.?someinfogainedfromcomparisons.基因敲除技術(shù)的應(yīng)用及前景1.

建立生物模型。在基因功能，代謝途徑等研究中模型生物的建立非常重要?；蚯贸夹g(shù)就常常用于建立某種特定基因缺失的生物模型，從而進(jìn)行相關(guān)的研究。這些模型可以是細(xì)胞，也可以是完整的動植物或微生物個體。最常見的是小鼠，家兔、豬、線蟲、酵母和擬南芥等的基因敲除模型也常見于報道。AnimalModelsofHuman

DiseaseCancerHypertensionHeartdiseaseDiabetesParkinson’sdiseaseAlzheimers’diseaseHuntingtondiseaseTay

SachsOsteogenesisimperfectaMusculardystrophyAmyotrophiclateralsclerosisInfertilitySicklecellanemiaAlpha-thalassemiaBeta-thalassemiaCysticfibrosisSCIDFamilialhypercholesterolemiaGrowthhormonedeficiencyPhenylketonuriaAtherosclerosisAsthmaAlcohol

preferenceDepression基因敲除技術(shù)的應(yīng)用及前景2.

疾病的分子機(jī)理研究和疾病的基因治療。通過基因敲除技術(shù)可以確定特定基因的性質(zhì)以及研究它對機(jī)體的影響。這無論是對了解疾病的根源或者是尋找基因治療的靶目標(biāo)都有重大的意義?；蚯贸夹g(shù)的應(yīng)用及前景3.

提供廉價的異種移植器官。眾所周知，器官來源稀少往往是人體器官移植的一大制約因素，而大量廉價的異種生物如豬等的器官卻不能用于人體。這是因為異源生物的基因會產(chǎn)生一些能引起人體強(qiáng)烈免疫排斥的異源分子，如果能將產(chǎn)生這些異源分子的基因敲除，那么動物的器官將能用于人體的疾病治療，這將為患者帶來具大的福音?；蚯贸夹g(shù)的應(yīng)用及前景4.

免疫學(xué)中的應(yīng)用。同異源器官移植相似，異源的抗體用于人體時或多或少會有一定的免疫排斥，使得人用抗體類藥物的生產(chǎn)和應(yīng)用受阻。而如果將動物免疫分子基因敲除，換以人的相應(yīng)基因，那么將產(chǎn)生人的抗體，從而解決人源抗體的生產(chǎn)問題?；蚯贸夹g(shù)的應(yīng)用及前景5.

改造生物、培育新的生物品種。細(xì)菌的基因工程技術(shù)是本世紀(jì)分子生物學(xué)史上的一個重大突破，而基因敲除技術(shù)則可能是遺傳工程中的另一重大飛躍。它為定向改造生物，培育新型生物提供了重要的技術(shù)支持?；蚯贸夹g(shù)的缺陷隨著基因敲除技術(shù)的發(fā)展，早期技術(shù)中的許多不足和缺陷都已經(jīng)解決，但基因敲除技術(shù)始終存在著一個難以克服的缺點，即敲掉一個基因并不一定就能獲知該基因的功能，其原因包括：1.

許多基因在功能上是冗余的，

敲掉一個在功能上冗余的基因，并不能造成容易識別的表型，因為基因家族的其他成員可以提供同樣的功能；基因敲除技術(shù)的缺陷對于某些必需基因，敲除后會造成細(xì)胞的致死性，也就無法對這些必需基因進(jìn)行相應(yīng)的研究了;很多疾病的發(fā)生都是多基因、多因素相互影響、相互作用的結(jié)果，單基因的敲除或突變的動物模型在這類疾病模型中應(yīng)用有限；有些基因敲除后的表型（智力）在動物模型上難以與人相比較。1.MolecularConsequencesofTargetingabcHSV-TKAAAAAAAAAAAAAAAAAAc dAAAAAAAAAAAAAAAAAAinsertions

[insertion/frame

shifttruncation

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUnstablemessagedegraded=no

proteinStable

message=mutant

proteinEmbryonicLethal-Now

What?-Conclusion:importantforembryonic

development-Furtherstudies-timedmatings-examinepupsatdifferent

stagesVLDLRGeneKnockout

Mice

Theverylowdensitylipoprotein(VLDL)receptorisamemberofthelowdensitylipoprotein(LDL)receptorfamilythatmediatesthebindinganduptakeof

VLDL.

Itssequenceis94%identicalinhumansandrabbitsand84%identicalinhumansandchickens,implyingaconservedfunction.ItshighlevelexpressioninmuscleandadiposetissuesuggestsaroleinVLDLtriacylglycerol

delivery.

Mutationsinthechickenhomologuecausefemalesterility,

owingto

impairedVLDLand

vitellogenin（卵黃生成素）

uptake

during

eggyolk

formation.VLDLRGeneKnockout

Mice

VLDLRgeneknockoutmiceofbothsexeswereviableandnormallyfertile.Plasmalevelsofcholesterol,triacylglycerol,andlipoproteinswerenormalwhenthemicewerefednormal,high-carbohydrate,orhigh-fatdiets.

Thesoleabnormalitydetectedwasamodestdecreaseinbodyweight,bodymassindex,andadiposetissuemassasdeterminedbytheweightsofepididymalfat

pads.

WeconcludethattheVLDLreceptorisnotrequiredforVLDLclearancefromplasmaorforovulationin

mice.

FrykmanPK.etal.Normalplasmalipoproteinsandfertilityingene-targetedmicehomozygousforadisruptioninthegeneencodingverylowdensitylipoproteinreceptor.ProcNatlAcadSciUSA.1995Aug29;

92(18):8453-8457.Themainreelinsignalingcascade(ApoER2and

VLDLR)Reelin(RELN)isalargesecretedextracellularmatrixglycoproteinthathelpsregulateprocessesofneuronalmigrationandpositioninginthedevelopingbrainbycontrollingcell–cellinteractions.VLDLRandApoER2GeneKnockout

Mice(C)Twenty-day-olddoubleknockoutmice(blackmice)aresmallerthannormallittermates(agoutianimalincenter).Doubleknockoutsfrequentlyshowmusclewasting(rightanimal)andprogressivehindlimb

paralysis.(DandE)Wild-typeanddoubleknockoutbrain(P20).Arrowspointtonormalandrudimentarycerebellumin(D)and(E),

respectively.TrommsdorffMetal,Reeler/Disabled-likedisruptionofneuronalmigrationinknockoutmicelackingtheVLDLreceptorandApoEreceptor2.Cell.1999,

11;97(6):689-701.VLDLRandApoER2GeneKnockout

Mice(A)Histoanatomyofhippocampusandcerebelluminwild-typeandmutantmice.Hematoxylin/eosinorsilver-stainedsagittalsectionsofhippocampusat4×(a,d,g,andj),cerebellumat2.53(b,e,h,andk),andcortex(c,f,i,andl)at20×relativemagnificationofP20wildtype(a–c),vldlr-/-(d–f),apoER2-/-(g–i),anddoubleknockout(j–l)areshown.Arrowsin(d)and(g)pointatthesplitintheCA1region,whichisalsoacharacteristicfeatureinreelerandmDab1-deficientmice.Thearrowin(k)pointstothesmallrimofdysplasticgranulecellsinthedoubleknockout.Thearrowin(l)pointstoaggregatedsilver-positiveneurofilamentsindoubleknockoutcortex(revealedbytheSiever-Mungermethod).Scalebar

in(b),1mm(2.5×),625mm(4×),

125mm

(20×).TrommsdorffMetal,.Cell.

1999,11;97(6):689-701.VLDLRandApoER2GeneKnockout

MiceInSituHybridizationandImmunohistochemicalDetectionofCalbindinandMAP-2inSagittalSectionsofWild-TypeandMutant20-Day-OldMice(a–d)InsituhybridizationsforCalbindin(darkfieldimage).(e–h)Immunohistochemicalstainingfor

Calbindin.Conclusion：theLDLreceptorgenefamilyandimplicatetheVLDLRandApoER2asessentialcomponentsoftheReelinpathwaythateitherdirectlyorindirectlyinteractwithextracellularReelinandcouplethesignaltomDab1inthe

cytosol.TrommsdorffM,etal.Cell.1999,

11;97(6):689-701.TayfunOzceliketal.Mutationsintheverylow-densitylipoproteinreceptorVLDLRcausecerebellarhypoplasiaandquadrupedallocomotioninhumans.PNAS.2008;105(11):4232

-4236.TissueSpecificCre-ExpressingMouse（條件性基因敲除法）Transgenic

MouseExpressesCreinasubsetof

tissues.Note:manytissue-specificCre-expressingmicehavebeenmade,morearebeing

made.CreTissueSpecific

PromoterTissueSpecificCre-ExpressingMouse12 3+CreTissueSpecific

Promoter13Targeted

MouseTransgenic

MouseMousewithknockoutONLYintissuesthatexpress

CremateloxP loxPTissueSpecificCre-ExpressingMouseIntroductionofPoint

MutationsintoMouse

GenomeHSV-TKNeomycin

R123*Neomycin

R123*loxPloxP123CreRecombinationin

TargetedEScells:Mutation

ConstructNeomycin

R123TransientCre

expression123**loxPloxPDominant-NegativeAllelesCanFunctionallyInhibitSome

Genes(a)Small(monomeric)GTPases(purple)areactivatedbytheirinteractionwithaguanine-nucleotideexchangefactor(GEF),whichcatalyzestheexchangeofGDPforGTP.Dominant-NegativeAllelesCanFunctionallyInhibitSome

Genes(b)Introductionofadominant-negativealleleofasmallGTPasegeneintoculturedcellsortransgenicanimalsleadstoexpressionofamutantGTPasethatbindstoandinactivatestheGEF.Asaresult,endogenouswild-typecopiesofthesamesmallGTPasearetrappedintheinactiveGDP-boundstate.Asingledominantnegativeallelethuscausesaloss-of-functionphenotypeinheterozygotessimilartothatseeninhomozygotescarryingtworecessiveloss-of-function

alleles.TissueSpecificRescueofKnockout

MousecDNAofKOed

geneTissueSpecific

PromoterTargeted

Mouse+Transgenic

MouseMousemissingproteinONLYintissuesthatdon’texpressthe

cDNA1423Neomycin

R3mateTemporalRegulationof

Cre:SteroidHormoneBindingDomain

FusionsERT-CreTissueSpecific

PromotercytoplasmnucleusER-CreERT-CrecytoplasmnucleusNo

hormone+

Tamoxifen基因敲入小鼠模型人源化小鼠模型條件性基因敲除（Conditional

Knockout）全基因敲除小鼠

（Conventional

Knockout）ROSA位點轉(zhuǎn)基因小鼠Transgenetic

technology轉(zhuǎn)基因技術(shù)采用基因轉(zhuǎn)移技術(shù)使目的基因整合入受精卵細(xì)胞或胚胎干細(xì)胞，然后將細(xì)胞導(dǎo)入動物子宮，使之發(fā)育成個體。Transgenetic

technologyForeignDNAinjectedintooneofthetwopronuclei(themaleandfemalehaploidnucleicontributedbytheparents)hasagoodchanceofbeingrandomlyintegratedintothechromosomesofthediploidzygote.Transgenetic

technologyBecauseatransgeneisintegratedintotherecipientgenomebynonhomologousrecombination,itdoesnotdisruptendogenous

genes.Transgenetic

technologyR.L.Brinster.etal.,1981,Cell27:223.Transgenetic

technologyZincFingerNucleases,

ZFNszinc

finger

-helix

-pleated

sheet

-turnzinc

ionsZincfinger

motifZincfingerand

DNAZincFingerNucleases,

ZFNsFokIisabacterialtypeIISrestrictionendonucleasewithoutfurthersequencespecificity.FokIcleavagemodel

(dimerization)ZincFingerNucleases,

ZFNsLeft

ZFPRight

ZFPZincFingerNuclease,

ZFNCleavage

domainThree-dimensionalmodelZFNdimerboundtoatypical,nonpalindromicDNA

targetJeffreyCMiller,etal.Animprovedzinc-fingernucleasearchitectureforhighlyspecificgenomeediting.NatureBiotechnology,2007,25,778-

785.ZincFingerNucleases,

ZFNs

ZincZincFingerFingerNuclease,Nuclease,

ZFNZFNAdouble-strandedbreak(DSB)intheDNAcanberepairedthroughtheprocessofhomologousrecombination(HDR)orthroughtheerror-proneprocess

ofnon-homologousend-joining(NHEJ).(LisaM.OttdeBruin,

2015)ZincFingerNucleases,

ZFNsZincFingerNuclease,

ZFN優(yōu)點：操作相對簡便、可在生殖細(xì)胞或受精卵內(nèi)進(jìn)行，實驗周期短。缺點：鋅指模序設(shè)計時特異性相對較低，陽性率較低。TranscriptionActivator-LikeEffectorNucleases,

TALENsTranscriptionActivator-LikeEffector

NucleasesTALENsbindDNAandgenerateaDSBupondimerizationoftheirFokI

domains.LisaM.OttdeBruin,etal.Novelgenome-editingtoolstomodelandcorrectprimaryimmunodeficiencies.Front.Immunol.2015,6:

250.ClusteredRegularlyInterspacedShortPalindromicRepeats(CRISPR)/CRISPRAssociated(Cas)System,CRISPR/

CasDiagramofthepossiblemechanismforCRISPRClusteredRegularlyInterspacedShortPalindromicRepeats(CRISPR)/CRISPRAssociated(Cas)System,CRISPR/

CasThekeystepsofCRISPR-Casimmunity.1)Adaptation:insertionofnewspacersintotheCRISPRlocus.2)Expression:transcriptionoftheCRISPRlocusandprocessingofCRISPR

RNA.3)Interference:detectionanddegradationofmobilegeneticelementsbyCRISPRRNAandCas

protein(s).CRISPR/Cas3

SystemInTypeIsystems,thepre-crRNAisprocessedbyCas5orCas6.DNAtargetinterferencerequiresCas3inadditiontoCascadeand

crRNA.TheCRISPR-Casimmunesystem:Biology,mechanismsandapplications.Biochi.2015,

1-10.CRISPR/Cas6

SystemTheTypeIIIsystemsalsouseCas6forcrRNAprocessing,butinadditionanunknownfactorperform3′end

trimming.Here,theTypeIIICsm/CmrcomplexisdrawnastargetingDNA,butRNAmayalsobe

targeted.TheCRISPR-Casimmunesystem:Biology,mechanismsandapplications.Biochi.2015,

1-10.CRISPR/Cas9

SystemTypeIIsystemsuseRNaseIIIandtracrRNAforcrRNAprocessingtogetherwithanunknownadditionalfactorthatperform50endtrimming.Cas9targetsDNAinacrRNA-guidedmanner.TheCRISPR-Casimmunesystem:Biology,mechanismsandapplications.Biochi.2015,

1-10.CRISPR/Cas9

Systemprotospaceradjacent

motifGenomeengineeringusingtheCRISPR–Cas9

systemFranciscoJ.etal.ApplicationsoftheCRISPR–Cas9systemincancer

biology.NatRveCancer.2015,15:387-395.CRISPR/Cas9

SystemInhumancells,DSBsmayberepairedthroughthedouble-strandbreakrepair(DSBR)pathwayorthenon-crossoversynthesis-dependentstrandannealing(SDSA)pathway.Bothcrossoverandnon-crossoverDSBRcan

occur.Inhumanembryos,repairofDSBsgeneratedbyCRISPR/Cas9occursmainlythroughNHEJ.IfHDRisutilized,thenon-crossoverpathwayis

preferred.CRISPR/Cas9

SystemSchematicrepresentationofzygoteinjectionwith

CRISPR/Cas9.CRISPR/Cas9

System

MITFprotein

disruptionSchematicoftheCas9/sgRNA-targetingsitesinpigmitf

lociXianlongWang,etal.EfficientCRISPR/Cas9-mediatedbiallelicgenedisruptionandsite-specificknockinafterrapidselectionofhighlyactivesgRNAsinpigs.SciRep.2015,Publishedonline:21

AugCRISPR/Cas9

System

MITFprotein

disruptionschematicdiagramofthetemplatesforinvitrotranscriptionusedtogeneratetheCas9mRNAand

sgRNA.CRISPR/Cas9

System

MITFprotein

disruptionOverviewandtimelineoftheexperiment.Beginningwithexperimentaldesign,assessmentofsgRNAmutagenesisefficienciescanbeachievedwithin10

days.CRISPR/Cas9

System

MITFprotein

disruptionNewbornwildtypepig(leftpanel)andpigletscarryingMitfgenemutation(right

panel).XianlongWang,etal.EfficientCRISPR/Cas9-mediatedbiallelicgenedisruptionandsite-specificknockinafterrapidselectionofhighlyactivesgRNAsinpigs.SciRep.2015,Publishedonline:21

AugCRISPR/Cas9

System

β-thalassemiamutatedgene

correctionCRISPR/Cas9couldeffectivelycleavethe

endogenousβ-globingene

(HBB).However,theefficiencyofhomologousrecombinationdirectedrepair(HDR)ofHBBwaslowandtheeditedembryoswere

mosaic.Off-targetcleavagewasalsoapparentinthese3PNzygotes.PupingLiang,etal.CRISPR/Cas9-mediatedgeneeditinginhumantripronuclearzygotes.ProteinCell.2015,

6(5):363–372CRISPR/Cas9

SystemApplicationgeneticallyengineeredmousemodels(GEMMs)

ANDnon-germlineGEMMs

(nGEMMs)FranciscoJ.etal.ApplicationsoftheCRISPR–Cas9systemincancerbiology.NatRveCancer.2015,15:

387-395.CRISPR/Cas9

SystemApplicationCRISPR-mediatedsomaticgenomeengineeringinvivocanbeusedtorapidlygeneratecohortsoftumour-bear