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Pnabio/./Reconstitutes to 5 mg/ml,6000 pmol (30 uM x 50 ul x 4 tubes)/Cas9 protein with NLS, high concentration/CP03/CP03

Cas9protein,incomplexwithguideRNA,offersbenefitforCRISPR/Cas9technologyoverplasmidormRNAtransfection.ThebiggestbenefitisthatyoucanmakeRNPcomplexthatisreadytoworkinvivo.Cas9proteinisshortlived(about24hoursofhalflifeincells),thusminimizetheoff-targeteffectsandmosaicism.YoucanalsoeasilytesttheactivityofguideRNAinvitrobyasimplecleavageassay.Inaddition,Cas9proteinisprettystable,andactivityconfirmationismucheasiercomparedtomRNA.Lastly,itisactiveinmorebroadrangeoforganismssuchasnematodes,insects,plants,andnon-typicalmodelsystemsasitdoesnotrequiretranscriptionortranslation.

Cas9proteinofferedbyPNABioisconjugatedwithNLS,anditisinjection/transfectionreadyform.

Cy3-Cas9canbeaneasyenrichmentofthecellsthataretransfectedwithCRISPR/Cas9complex.

Invitrocleavageassay

Cas9 protein NLS cleavage assay with sgRNA

PCRproductwasincubatedwithvariousamountsofCas9proteinandrunonthegel.InvitrocleavageassayisasimplewaytochecktheactivityofsgRNA.

TransfectionofRNPcomplex

Cas9 protein NLS cleavage assay with sgRNA

RNPcomplexofsgRNAandvariousamountofCas9proteinwastransfectedintoNIH3T3cellsbyelectropration(Neon)orbylipofectamine(LF2000).

InjectionofRNPcomplex

Cas9 protein NLS cleavage assay with sgRNA

Formouseembryoinjection,RNPcomplexof60ng/ulofCas9proteinand15ng/uleachoftwosgRNAswasinjectedintomouseembryo.ThetailDNAwaspreppedfromthepupsandsubjecttoT7E1assay.GreyrepresentsWT,greenforlargedeletion(#2,3,4,8),redforsmallin/del(#5,6,7),blueforhomozygoussmallin/del(#9,10).

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