Gene Knockout Mediated byCRISPRCas92hitKO 50文档格式.docx
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Gene Knockout Mediated byCRISPRCas92hitKO 50文档格式.docx
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Toachievehigherknockoutefficiency,vectorcarryingonecas9proteinandtwoguideRNAexpressingcassettesisgenerated.Inthisprotocol,threeplasmidsareinvolved.PX458MandPX459Mcontaincas9andoneGuideRNAexpressingcassette.AdditionallytheMCSisfortheinsertingofthesecondGuideRNAexpressingcassette.EZ-GuideXHisforthepreparationofthesecondguideRNAexpressingcassette.
PX458McontainsEGFPforfluorescentmarking;
PX459Mcontainspuromycinresistantgenefordrugselection.
Figure3.MapforPX458M
Figure4.MapforPX459M
HindIII
XhoI
Figure5.MapforEZ-GuideXH
PX458MandPX459MarereconstructedfromPX458andPX459plasmidsfromZhangFeng’slabinMITthroughaddgene.(PX458andPX459plasmidsaregiftfromProf.XiQiaoran’slab)ThemainalterationisadditionofonemultiplecloningsiteforthesecondguideRNAexpressingcassette.
EZ-GuideXHisgeneratedbasedonEZ-Tcloningvector.ThemainpurposeofthisplasmidisforpreparationofthesecondguideRNA.
AssemblyofGuideRNA
AccordingtoZhangFenglab’sprotocol,guideRNAcanbeeasilyassembledthroughaningeniousstrategy.ThisstrategyisshowninFigure6.WhenGuideRNAscaffoldisdigestedbyBbsI,anannealedduplexwithspecificstickingends(as20Nucleotides’target)willbeligatedinthisplasmidthroughT4DNAligase.
Figure6.StrategyforguideRNAassembly
………………………………………………………………………………………………………………………...
Procedure
I.PickCas9targetwithminimaloff-targetspotentials
a.AvoidtoomuchTsinthe3’armend.
b.Todecreasepotentialoff-targeteffect,thecandidatetargetshouldshowlowpossibilitythatmatchesunexpectedsitesinthegenome.Thecriteriaincludes:
1)Themismatchesmustexceed2nucleotides.
2)Especially,thewell-matchinginthe3’armendshouldbeavoided.
3)Avoidthepotentialoff-targetsfollowedbyNGGmotif.
AlthoughCRISPR-Cas9systemisahighefficientgenomeeditingtool,highrateofoff-targetseffectisthemajorproblembecauseofthenonspecificrecognitionoftargets.SoitisnecessarytopickoutCas9targetswithminimaloff-targetpotentials.
Fortunately,somesoftwareandonlinetoolsaredevelopedtosolvethisproblemthroughcalculatingthepotentialsofoff-targetsforcertaintarget.
Websitefortargetprediction
http:
//crispr.mit.edu/.Thebriefmanualisasbelow(Figure7.)
1.Inputnomorethan250nts’genomicDNAsequencetotheinputbox,andpickthecorrespondingspecies.Thenclickbutton‘submit’.
2.Clickbutton‘Guides&
offtargets’.
3.Choosetargetswhosescores>
90.Thetargetscanbeinbothorientations.
Figure7.SubmissionofpredictionofguideRNAtarget
II.PickapairofCas9targetstodeletetheobjectiveDNAfragment
TodeleteagenomicDNAfragment,twotargetsrecognizedbyguideRNAareindispensable.Accordingtoprevioustest,aslongas1Mbpssequencecanbedeletedefficiently.However,fragmentdeletionof<
10kbsshouldbefineandefficientenough.Thecriteriaofchoiceontargetpairisasfollowed,(Figure8andFigure9aretwoexamples)
a.Iftheinterestedgeneisshort(about10kbs,oralittlelarger),justdeletethewholegeneincludingpromoterregion.
b.Iftheinterestedgeneislarge,deletingthemostimportantexonorexonsshouldbeagoodchoice.
c.Iftheinterestedgeneislarge,analternativeisdeletingapartofthegeneincludingwholepromoterregion
d.Iftheinterestedgenewasknockoutbytraditionalstrategypreviously,justdeletethesameregion.
Figure8.Cdkn2aCas9-targeting
Figure9.PTENCas9-targeting
III.ConstructCRISPR-Cas9vectorcarryingtwoGuideRNAexpressingcassettes
Figure10.Overviewofconstruction
1.Synthesistargetoligos.
Oligo-Forward:
CACC-(N)20
Oligo-Reverse:
AAAC-(N)20R
Ifthefirstnucleotideofthetargetsequenceisnot‘G’,justaddone‘G’tothe5’end.Theoligosbecome
Forward:
CACC-G(N)20
Reverse:
AAAC-(N)20RC
2.Annealing
Oligo-Forward(100uM)
1ul
Oligo-Reverse(100uM)
10xT4ligasebuffer
T4PNK
H2O
6ul
Totalvolume
10ul
Onthermalcyclerwithheatlid.
37℃,30min
95℃,5min
Rampdownto25℃at0.1℃/sec.(whenstep25℃ishighlighted,clickoption,setRampas0.1℃/sec)
25℃,5min
4℃,5min
Putonice,orkeepin-20℃freezerforstorage.
3.DigestPx458M(orPX459M)andEZ-Guide-XHwithBbsI
Plasmid
6-10ug
BbsI
2ul
10xbuffer
5ul
Upto50ul
50ul
PurifytheenzymedigestedproductwithPCRproductpurificationkit,andmakesurethattheconcentrationismorethan50ng/ul.
4.Ligation
Dilutetheannealedtargetoligos200timesforfurtherligation
Dilutedannealedoligos
Digestedplasmid
100ng
T4ligase
Upto10ul
Ligateformorethan2hoursat16℃.(Overnightisbetter.)
5.Transformationandidentificationofpositiveclones
Picksingleclonesandcultureforseveralhoursin37℃incubator,andperformPCRtoidentifythepositiveclones.
ForPx458MandPx459M,useOligo-ForwardandCAG-Rasprimers;
forEZ-GuideXH,useOligo-ForwardandM13Fasprimers.
6.DigestGuideRNATarget2expressingcassetteintheEZ-GuideXHandPX458M-Target1(orPX459M-Target1)
EZ-GuideXH-Target2
10ug
XhoI
10xbufferR
PX458M-Target1
7.Gelextractionandpurification
ForEZ-GuideXH-Target2,cutandpurifythesmallerDNAbandabout360bps;
forthePX458M-Target1,cuttheuniquemainbandabout3300bps.
8.Ligation
DigestedPX458M-Target1
DigestedTarget2
20ng
Ligateovernightat16℃.
9.Transformationandidentificationofpositiveclones
UseOligo-Forward(target1)andCAG-Rasprimers,andthePCRproductisabout750bps.
CAG-screenR
GTACTGGGCACAATGCCAG
10.Plasmidextractionandsequencingvalidation.
Culturethepositiveclonein10mlAmp+LBmedium,andextractplasmid.
UseCAG-Rassequencingprimer.
IV.Assessknockoutefficiencyin293FTcells(orothercells)
1.TransfectPX458MplasmidcarryingGuideRNAexpressingcassettesin293FTcellsfor36-48hours.Formousegenes,MEF,3T3andsomeothereasy-transfectcellscanbeused.
2.DetecttheGFPFluorescencethroughfluorescentmicroscopyorexaminethetransfectionefficiencythroughFlowcytometer.
3.ExtractgenomicDNAfromtransfectedcells.AndperformgenotypingwiththesegDNAsamples.IfthemutantPCRbandisobviousandstrongenough,thepairofGuideRNAscanberegardedasefficient.Figure11showthereglularstrategyforknockoutgenotyping;
andFigure12showtheexampleofPPM1AKnockout.
Figure11.Diagramofgenotyping
Wtband
Mutband
Figure12.GenotypingofPPM1Aknockout
V.Knockoutininterestedcells
Theoretically,anycellstransfectedwithplasmidshouldbeKOcells.However,togetpureKOclones,singleclonesculturearenecessary.Cultureofsingleclonesisalittledifficultforsomecelltypes.Toimprovetheexpansionofsinglecells,gelatinpre-coatingandconditionalmediumcanincreasethesurvivalrateofsinglecells.
1.Platethecellsin6-wellplateonedaybefore,andmakesurethecellconfluencyreachabout70-80%.
2.Transfecttheinterestedcellswithvalidatedpx459Mbasedplasmidandculturefor24hours.
3.Treatthetransfectedcellswith2ug/mlpuromycinfor3or4days.Setonemockun-transfectedwellascontrol.
Cautions!
:
beforetreatment,apilotexperimentisneededtoexploretheoptimizedpuromycinconcentration.
4.Withdrawthepuromycin,andculturethecellswith20%FBSmedium.
5.Severaldayslater,ifindependentclonesexpand,picktheclonesandtransfertheminto24-wellplateforculture.Theremainedcellskeepgrowinguntilabout50%cellconfluency.
6.Whenthecellsin24-wellplatereachmorethan30%confluency,trypsin-digestthesewells,takeou
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