PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-gRNA-Pac1
ProductCode:OG3518
Size(bp):4723bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
ThisplasmidisdesignedtoexpressaguideRNAofchoiceinmammaliancells.ExpressionisdrivenbyaU6promoter.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.ForthisreasonwestockarangeofotherpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.TheguideRNAisterminatedatastringofTresiduesthatterminatesIIIRNApolymerase.
Cloning:
Cloninginagene:Thisplasmidhasbeendesignedtobecompatiblewitharangeofcloningtechniques.Themultiplecloningsitecontainsarangeofstandardcommonlyusedrestrictionsitesforcloning.UsingthesesitesgenescanbeinsertedusingstandardcloningmethodswithDNAligase.Othermethodssuchasligaseindependentcloning(LIC)GibsonAssemblyInFusionHDorSeamlessGeneArtcanalsobeusedandbecauseallofourplasmidsarebasedonthesamebackbonethesamemethodcanbeusedforcloningintoallofourcataloguevectors.
Werecommendselectingatargetsequencetowardsthe5’endofthegenetobedisrupted.Therearevariousonlinetoolsavailabletohelpchoosethebesttargetsitedependingonyourmodelorganism.Forinstance:http://www.rgenome.net/cas-designer/orhttps://chopchop.rc.fas.harvard.edu/
Chooseagenomictargetsequenceoftheform:(examplesequenceingreen,thesequenceNGGinredmustexistatthegenometargetcutsite,butitwillnotbeintheguideRNA)
5’ TGCTTAAAATACTCCGCCGNGG 3’
(19bp) PAMsite
Designtheforwardoligobyaddingthefollowingends(darkblue)toyour19bptargetsequence:
5’ AAACACCGTGCTTAAAATACTCCGCCGGTTTTAGAG 3’
Designthereverseprimerbyreversecomplementingyour19bptargetsequence(orange)
5’ CGGCGGAGTATTTTAAGCA 3’
andthenaddthefollowingends(lightblue):
5’ CTAGCTCTAAAACCGGCGGAGTATTTTAAGCACGGT 3’
Whentheyareannealed,theoligonucleotidesyouhavedesignedwillannealtoformadsDNAoligowithoverhangsthatmatchtheoverhangsoftheBsaIcutvector.
5’ AAACACCGTGCTTAAAATACTCCGCCGGTTTTAGAG 3’
3’ TGGCACGAATTTTATGAGGCGGCCAAAATCTCGATC 5’
The5’terminioftheoligosshouldbephosphorylatedeitherbyselectingthatoptionwhenorderingthemorbytreatmentwithPNK(linktoOGprotocol).
ByusingaspeciallydesignedadapterDNAitispossibletoclonetwogRNAtargetsequencesintandem.Thiscanbedonebydesigningoligosinthefollowingway:
gDNA1
Designthefollowingforwardandreverseoligosbyaddingthefollowingendsequences(lightblue)toyourgenomictargetsequence(green).
Forwardprimer:
5’ AAACACCGTGCTTAAAATACTCCGCCGGTTT 3’
Reverseprimer:
5’ TCTAAAACCGGCGGAGTATTTTAAGCACGGT 3’
Result:
5’ AAACACCGTGCTTAAAATACTCCGCCGGTTT 3’
3’ TGGCACGAATTTTATGAGGCGGCCAAAATCT 5’
gDNA2
Designthefollowingforwardandreverseoligosbyaddingthefollowingendsequences(lightblue)toyourgenomictargetsequence(purple).
Forwardprimer:
5’ ACCGTCCCAGTGGGTAACAATCCGTTTTAGAG 3’
Reverseprimer:
5’CTAGCTCTAAAACGGATTGTTACCCACTGGGA 3’
Result:
5’ ACCGTCCCAGTGGGTAACAATCCGTTTTAGAG 3’
3’AGGGTCACCCATTGTTAGGCAAAATCTCGATC 5’
Byusingthelessefficientbutmoreprecisehomologydirectedrepair(HDR)pathway,itispossibletomakeaspecificeditofthegenomicDNA.ThisrequiresaDNArepairtemplate.Thelengthoftherepairtemplatedependsontheapplicationandhowlargeamodificationisbeingmade,butitcanbeasinglestrandedoligonucleotide,doublestrandedoligonucleotideoradoublestrandeddonorplasmid.Evenrelativelylargesequences(severalkbp)suchasfluorescentorselectableMarkerscanbeinsertedusingthismethod.Therepairtemplatemustcontainthedesiredmutation.ItmustalsonotcontainthePAMsiteusedbythegRNA,otherwisetherepairtemplatemayitselfbecomeatargetforCas9.DuetothelowefficiencyofHDRcomparedtoNHEJ,generallyonly10%ofmodifiedallelesmaycontainthedesiredmutation.Therefore,itisimportanttoconfirmthepresenceofthedesiredmutationexperimentally.
Thedonorvectormustcontain:
1.Thedesiredmutation.
2.Aselectableand/orfluorescentmarker.
3.Armsofhomologyupstreamanddownstreamtheexpressioncassetteeachofwhichshouldcontainapproximately800bpofhomologoussequencetothegenomicregionsurroundingthetargetsite(figure2).
Figure2:Insertionofasyntheticconstructbyhomologousrecombination.Cas9cleavesthegenomicDNA(blue)atthetargetsiteleADIngtoadoublestrandbreak.Ifarepairtemplateissuppliedonadonorvector(green),thebreakcanberepairedbyhomologousrecombination.Inthisexamplethedonorvectorisbeingusedtoknock-inanexpressioncassette.Thecassetteisflankedby800bpofDNAsequencewithhomologytothegenomeoneithersideofthetargetsite.Theactualsitesofrecombination(redcrosses)mayvarydependingonthepositionoftheHolidayjunctionswhentheyareresolved.
ThearmsofhomologyshouldbedesignedtoflanktheCas9cleavagesite(2-4bpupstreamthePAMsite)ascloselyaspossible(lessthan10bp;figure3).
Figure3:Armsofhomology.NotethatthedonorvectordoesnotcontainthecompletegRNAtargetsequence.Eacharmofhomologybegins1bpawayfromthelikelybreaksite.
ItisalsoimportantthatthedonorvectordoesnotcontainthecompletegRNAtargetsequenceotherwisethedonorvectorwouldbecleavedbyCas9.Thiscanbedonebyplacingtheinsertinthemiddleofthetargetsequence(asinfigure3)orbyalteringthePAMsequenceinthedonorvector.IfthePAMsiteiswithinacodingregion,caremustbetakentomakesurethealterationisasilentmutation.
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Multiplecloningsitenotes:ThereareafewimportantsiteswithintheMCS.TheseincludetheNcoIsitetheXbaIsiteandtheBsgIandBseRIsites.TheNcoIsitecontainsastartcodonthatisimmediatelydownstreamofbothaKozakandShine-Dalgarnoribosomalbindingsite.Theseallowforoptimalpositioningofgeneswhenthestartcodonisplacedinthislocation.IfthisisnotrequiredandyouwishtouseadownstreamsiteforgenecloningyoucanremovetheNcoIsitebycleavingtheplasmidwithKpnI.
BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsites.Thisallowsthemtocuttheupstreamstopcodoninthegeneinthisplasmidregardlessofthegenesequence.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastProplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
