The CRISPR/Cas9 Mediated Gene Activation System By Tet-on/off

CRISPR-on is an RNA guided system which can activate multiple endogenous genes transcription.In modified CRISPR/Cas9 system,its two catalytic domains RuvC and HNH have been inactivated,and the transcription activating domains such as VP64,HSF1 and p65 have been fused with Cas9 protein.The effective CRISPR/Cas9 system needs both Cas9 protein and short guide RNA(sgRNA).In our study,we inserted the sgRNA to Tet(Tetracycline)inducible vectors via simple clone strategy,while changed nothing with the dCas9(inactivation of Cas9 nuclease)and transcription activators.Besides the CRISPR-on transcription activation,we achieved a dual regulation through manipulating sgRNA expression with Tet-on/off.We designed a sgRNA scaffold which contained a conservative structure sequence and 20bp changeable sequenced complementary with target genes,and combined it with Tet regulatory plasmids(pLVCT-rtTR-KRAB-2SM2&pLVCT-tTR-KRAB).Since lentiviral genetic sequence can be integrated to host genome and exist in long-term.We assembled the Tet induced sgRNA,Cas9 and transcription activators into lentivirus,and infected them to A549 cells(human lung adenocarcinoma cells)simultaneously.We screened A549 stable cell line containing Cas9 protein and transcription activators by using Puromycin and G418(Neomycin).The subsequent step was to infect the cell with sgRNAs.Window of-200bp to+1 upstream the transcription start site(TSS)is the sgRNA activation target site.According to the guideline,we designed and assembled lentivirus containing sgRNAs targeting human growth hormone(GH),insulin(INS)and uncoupling protein(UCPI),and infected the cell line with them.After Doxycycline inducing,we tested the genetic profile with Q-PCR(Quantity PCR)and validated that the CRISPR-on can upregUlate some genes expression.We have screened the A549 stable cell line expressing Cas9 protein and transcription activators,which could enhance the efficiency of activation and shorten the experimental cycle.At the same time,the Tet regulation system highlights the features of low basal leakage,high sensitivity and tight controllability.Above all,we could achieve the purpose of controlling gene expression timely and effectively through addition or withdrawing drugs.