Content:20µgOverview
Stem
MACS™Cas9NucleasemRNAencodestheCas9nucleasefrom
Streptomycespyogenesfortransfection.
Details
Backgroundinformation
Cas9originatesfromtheCRISPR(ClusteredRegularlyInterspersedPalindromicRepeats)adaptiveimmunitysystemofStreptococcuspyogenes.
Cas9nucleasecanbeusedtointroducesite-directeddoublestrandbreaksintothegenomicDNA.Thesewillsubsequentlyberepairedthroughoneoftworepairpathways:non-homologousendjoining(NHEJ)orhomologydirectedrepair(HDR).NHEJrepairiserrorproneandwillfrequentlyresultinnucleotidedeletionsorinsertions(indel).Ifsuchinsertions/deletionsresultinaframeshiftorintroductionofaprematurestopcodon,theywilldisrupttheopenre
ADIngframeofthetargetgene.Therefore,thisapproachcanbeusedtogenerateageneknockout.
Alternatively,theHDRpathwaycanbeusedtointroducespecificchangesintothegeneofinterest.ByprovidingaDNArepairtemplatethatcontainsthedesiredmodificationflankedbyregionsthatarehomologoustothegenomictargetsequence,HDRenablesgenomicintegrationofeditedtargetsequence.
ThepositionoftheCas9cleavagesiteisdeterminedbytwofactors:thetargetsequencerecognizedbytheguideRNAandthepresenceofaProtospacerAdjacentMotif(PAM,inthecaseofStreptococcuspyogenesCas9thenucleotidesequenceNGG).TheCas9/guideRNAcomplexwillrecognizetheconjunctionofbothsequenceelementsandgenerateadoublestrandbreak3basepairsupstreamofthePAM.
Detailedprocedure
Forsatisfactorytransfectionresults,useatransfectionprotocoloptimizedforyourspecificcelltype.StemMACS™eGFPmRNAorStemMACS™NucleareGFPmRNAalloweasyevaluationoftransfectionefficiencyandarerecommendedaspositivecontrols.
Qualitydescription
StemMACS™mRNAsarehighlypure,invitro-transcribedmRNAsthathavebeencarefullyoptimizedtoensurehighlevelexpressionaftertransfection.PolyadenylationandenzymaticcapmimicafullyprocessedmaturemRNAandensureefficienttranslation.Incorporationofthemodifiednucleotidespseudouridineand5-methylcytidinereducestheinnateantiviralresponsetosingle-strandedmRNA.Robustexpressionoftheencodedfactoraftertransfectionhasbeenverifiedbyimmunofluorescenceorflowcytometry.
StemMACS™mRNAsarelyophilizedfroma0.2µMfilteredsolutionforextendedshelf-life.