A Non-cloning Method For Efficiently Inducing Gene Expression Using CRISPR/Cas9 Technology

Objective: To establish a simple and efficient activation of endogenous gene expression studies using CRISPR/Cas9 technology.Methods: The expression of the endogenous gene is site-specifically induced by a synthetic sgRNA expression cassette to direct a nuclease-null Cas9 complex comprising a transcriptional activator(VP64,p65 and Rta).We designed the SpCas9 sgRNA expression cassette and the SaCas9 sgRNA expression cassette to test two CRISPR activation systems(dSpCas9-VPR,dSaCas9-VPR)by inducing multiple genes in human and rat cells.The transfection efficiency of different cells was tested by pmaxGFP transfection.Gene activation was detected by CRISPR/Cas9 activators.The relative expression of the target gene was compared by qPCR analysis.The gene expression induced by transcription factors was detected by immunocytochemistry analysis.Results: Both CRISPR/Cas9 activators can efficiently induce activation of six different neural development genes(CRX,RORB,RAX,OTX2,ASCL1 and NEUROD1)in human cells,while inducing activation of three different genes in rat cells(Ascl1,Neurod1,Nrl)showed more variable and less-efficient levels of gene induction.Conclusions: This study provides a simple method for the efficient activation of endogenous gene expression using CRISPR/Cas9 activators,which can be used in rapid workflows to facilitate gain-of-function studies for a range of molecular and cell biology disciplines.