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.
InvitrocleavageassayPCRproductwasincubatedwithvariousamountsofCas9proteinandrunonthegel.InvitrocleavageassayisasimplewaytochecktheactivityofsgRNA.
TransfectionofRNPcomplexRNPcomplexofsgRNAandvariousamountofCas9proteinwastransfectedintoNIH3T3cellsbyelectropration(Neon)orbylipofectamine(LF2000).
InjectionofRNPcomplexFormouseembryoinjection,RNPcomplexof60ng/ulofCas9proteinand15ng/uleachoftwosgRNAswasinjectedintomouseembryo.ThetailDNAwaspreppedfromthepupsandsubjecttoT7E1assay.GreyrepresentsWT,greenforlargedeletion(#2,3,4,8),redforsmallin/del(#5,6,7),blueforhomozygoussmallin/del(#9,10).
SouthernBiotech/Goat Anti-Human Lambda-Alexa Fluor® 555/2070-32/1.0 mg
vectorlabs/Biotinylated Aleuria Aurantia Lectin (AAL)/B-1395/1 mg
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Mouse IgG)/PK-6102/1 kit
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Universal)/PK-6200/1 kit
vectorlabs/Unconjugated Musa Paradisiaca (Banana) Lectin (BanLec)/L-1410/5 mg
GiottoBiotech/Calmodulin N60D/5 mg/G02CLM60cn