Description: TheEnGensgRNASynthesisKit,
S.pyogenes providesasimpleandquickmethodfortranscribinghighyieldsofsgRNAinasingle30minutereaction,usingthesuppliedreagentsandtarget-specificDNAoligosdesignedbytheuser.
Innature,
S.pyogenes Cas9isprogrammedwithtwoseparateRNAs,thecrRNAandtracrRNA.ThecrRNA,orCRISPRRNAsequencecontainsapproximately20nucleotidesofhomologycomplementarytothestrandofDNAoppositeandupstreamofaPAM(ProtospacerAdjacentMotif)(NGG)sequence.ThetracrRNA,ortransactivatingcrRNA,containspartialcomplementarysequencetothecrRNAaswellasthesequenceandsecondarystructurethatisrecognizedbyCas9.ThesesequenceshavebeenadaptedforuseinthelabbycombiningthetracrRNAandcrRNAintoonelongsingleguideRNA(sgRNA)(1)speciescapableofcomplexingwithCas9torecognizeandcleavethetargetDNA.
TheEnGensgRNASynthesisKit,
S.pyogenes combinesan
S. pyogenes Cas9-specificScaffoldOligo(includedintheEnGen2XsgRNAReactionMix)thatispartiallycomplementarytothetarget-specificoligosdesignedbytheuser.ThetwooligosannealattheoverlappingregionandarefilledinbytheDNApolymerase,creatingadouble-strandedDNA(dsDNA)templatefortranscription.SynthesisofthedsDNAtemplateandtranscriptionofRNAoccurinasinglereaction,resultinginthegenerationofafunctionalsgRNA.
1. Jinek,M.etal.(2012)
Science 816–821.PubMedID:22745249.
Figure1:GeneralworkflowfortheEnGensgRNASynthesisKit,S.pyogenes .Figure2:EnGensgRNASynthesisKitOverview A.Thetarget-specificoligocontainstheT7promotersequence,~20nucleotidesoftarget-specificsequenceanda14nucleotideoverlapsequencecomplementarytotheS.pyogenesCas9ScaffoldOligosuppliedinthereactionmix.Target-specificoligos(orEnGensgRNAControlOligo,S.pyogenes)aremixedwiththeEnGen2XsgRNAReactionMix(NTPs,dNTPs,S.pyogenesCas9ScaffoldOligo)andtheEnGensgRNAEnzymeMix(DNAandRNApolymerases)atroomtemperature.B.At37°Cthetwooligosannealatthe14nucleotideoverlapregionofcomplementarity.C.TheDNApolymeraseextendsbotholigosfromtheir3´endscreatingadouble-strandedDNAtemplate.D.TheRNApolymeraserecognizesthedouble-strandedDNAoftheT7promoterandinitiatestranscription.TheresultingsgRNAcontainsthetarget-specific/crRNAsequenceaswellasthetracrRNA.Allstepsoccurinasinglereactionduringa30minuteincubationat37°C. Figure3:ExamplesofsgRNAssynthesizedusingtheEnGensgRNASynthesisKit,S.pyogenes andmultipledifferenttarget-specificoligosincludingtheEnGensgRNAControlOligo,S.pyogenes . RNAwasrununderdenaturingconditionsona10%NovexTBE-Ureagelandpost-stainedwithSYBRGold.KitComponents Thefollowingreagentsaresuppliedwiththisproduct:
Storeat(°C) Concentration EnGen®sgRNAEnzymeMix -20 EnGen®2XsgRNAReactionMix,S.pyogenes -20 DNaseI(RNase-free) -20 2units/μl EnGen®sgRNAControlOligo,S.pyogenes -20
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