PlasmidInfo:

PlasmidPurpose:

Plasmidvectorforexpressingwild-typeStreptococcuspyogenesCas9proteininmammalincellsfromaCMVpromoterexpressioncassetteinthemainpSFMCS.AlsocontainsacassetteforexpressingoneortwoguideRNAs.

ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.ForthisreasonwestockarangeofotherpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.

SequenceandMap:

OtherInfo:

Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.TheguideRNAisterminatedatastringofTresiduesthatterminatesIIIRNApolymerase.

Cloning:

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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BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsites.Thisallowsthemtocuttheupstreamstopcodoninthegeneinthisplasmidregardlessofthegenesequence.

IPStatus:

ThisproductispartofourSnapFastProplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere