CRISPR/dCas9 Based Rapamycin-modulated Transcriptional Activation System

Objective:Transcriptional activation targeting specific genomic sites is helpful in understanding the complex gene networks,discovering the regulatory factors upstream and downstream,creating animal models,treating illness,medical and industrial applications.Through constructing rapamycin-controlled transcriptional regulatory systems,we can regulate the expression level of targeting genes both temporally and spatially.We can also build relating animal models.By inducing up-regulation of tumor suppressor genes,the system offers us the opportunity in treating malignant diseases and laying the foundation of clinical translational research.Methods:sgRNAs targeting multiple genomic sites,PSL690-dCas9-FKBP plasmid(the anchor)and the PSL690-FRB-effector domain plasmid(the activator)were designed and synthesized.sgRNA,anchor plasmid and activator plasmid were co-transfected into 293FT cells.Upon adding rapamycin,FKBP bind with FRB,transcriptional factor VP64 was dragged by sgRNA to promote mRNA expression of downstream genes.Cellular RNA was extracted,reverse-transcribed.Realtime-PCR was applied to detect upregulation of specific genes.Rapamycin concentration gradients(0 nM、100 nM、200 nM、500 nM、1000 nM and 2000 nM)were added and cck-8 was used to detect toxicity of rapamycin to 293FT cells on day 1,2,3 and 4.We optimized the FRB-VP64 and replaced the VP64 domain with p65,p65-VP64 and VP64-VP64.The activator plasmids were transfected into 293FT cells,and western blotting was used to detect basic expression of these plasmids.Multiple sgRNAs targeting other genes were designed and transfected into HeLa cells.Realtime-PCR was used to detect up-regulation of these genes in HeLa cells.Results:VEGFA、IL1RN and ASCL1 mRNA expression levels were up-regulated using dCas9-FKBP and FRB-VP64 transcriptional activation system.Concentration gradients of rapamycin(100 nM,200 nM,500 nM,1000 nM,2000 nM)revealed no growth inhibition in 293FT cells detected using CCK-8 assay.dCas9-FKBP plasmid was paired with FRB-VP64,FRB-p65,FRB-p65-VP64 and FRB-VP64-VP64 plasmids respectively to compare the availability of transcriptional activation.dCas9-FKBP paired with FRB-VP64-VP64 showed the highest ability in transcriptional activation using Realtime-PCR.And the system also upregulated mRNA of bakl、Caspase9、bim and Bad genes in HeLa cells.Conclusion:We constructed anchor plasmid-dCas9-FKB,activator plasmid FRB-VP64,FRB-p65,FRB-p65-VP64,FRB-VP64-VP64 to function as transcriptional activators.The anchor+activator system up-regulated mRNA levels specifically.Of all the systems,the dCas9-FKBP paired with FRB-VP64-VP64 showed the highest activity in transcriptional activation.Western blotting demonstrated that mRNA upregulation was not related to basic expression of anchor and activator plasmids.Further experiments evaluated applicability of dCas9-FKBP and FRB-VP64-VP64system in HeLa cells.