Generation Of Specific Gene Mutations By Expressing The SgRNA Of The CRISPR System From The RNA Polymerase Ⅱ Promoters

Recently, the CRISPR/Cas9 system is emerging as a powerful tool for genome editing and genetic screening, and holds great promise for biomedical applications in disease modeling and gene therapy by in vivo genome editing. However, the applications of C RISPR/Cas9 system still face some technical hurdles, one of which is to harness the gene editing in a precisely controlled manner. Of the two-component CRISPR/Cas9 system for genome editing, the Cas9 is a fixed genome-cutting component expressed from the RNA polymerase Ⅱ(pol Ⅱ) promoter that can drive tissue-specific gene expression; while the single-guide RNA(sg RNA) is a changeable genome-guiding component expressed from RNA polymerase Ⅲ(pol Ⅲ) promoter that usually drives the ubiquitous expression of "housekeeping" genes in all tissues. Therefore, to express the sgRNA in a tissue-specific manner can provide a convenient approach to tissue-specific gene mutations. Here, we reconstructed the sgRNA to enable its expression from the pol Ⅱ promoters, and further achieved cell-type specific gene mutations via the modified CRISPR/Cas9 system by using cell-type specific pol Ⅱ promoters-driving sgRNA.To generate pol Ⅱ promoter-driving sg RNAs, we constructed a microRNA-shRNA embedded sg RNA(mi Rsh-sg RNA) cassette that could express the small RNA from pol Ⅱ promoter into the 3’-untranslational region(UTR) of the Ds Red reporter gene. To test whether functional sgRNA can be efficiently derived from the pol Ⅱ promoter-driving mi Rsh-sgRNA cassette, we transfected mouse embryonic fibroblasts(MEFs) and mouse embryonic stem cells(mESCs) with constitutiveEF1 a promoter-driving miRsh-sgp53 expression vector and Cas9-P2A-EGFP expression vector. GFP and Ds Red double positive cells were sorted by fluorescence-activated cell sorting(FACS) two days after transfection for further analysis the T7EN1 cleavage assay of these cells showed that constitutive EF1 a promoter-driving miRsh-sgp53 can guide Cas9 for producing double strand breaks(DSBs) in p53 gene in both MEFs and mESCs. Further, to examine whether the mi Rsh-sgRNA cassette can produce gene mutation in a cell type-specific manner, we constructed an expression vector using the embryonic stem cell-specific mouse Oct4 gene promoter(mOct4P) to express the mi Rsh-sgp53 cassette. Two days after transfection of MEFs and mESCs with this vector and the EF1 a promoter-driving Cas9-P2A-EGFP vector, GFP and Ds Reddouble-positive mESCs were observed, but only GFP positive MEFs were observed, indicating the cell-type-specific expression of the sgRNA. We sorted GFP and DsRed double positive mESCs and GFP positive MEFs by FACS for further analysis. T7EN1 cleavage assay and Sanger sequencing were performed, and p53 gene mutation was only detected in the mESCs but not in the MEFs.Moreover, The T7EN1 assay and Sanger sequencing revealed that no potential off target site we tested was mutated by miRsh-sgp53 in all the examined cells.