CRISPR/Cas9-mediated Insertation Of Inducible Cas9 Expressing Cassette Targeting At The AAVS1 Locus In Human Cells

AAVS1 locus is a specific sequence located in the first introns of human gene PPP1R12C. It has the advantages of stability and not affecting other gene transcription even though exogenous gene is inserted into it. In recent years. CRISPR/Cas9, a new gene editing technology appears. This technology needs a 160kd Cas9 protein and a small fragment sgRNA in cells when it does the gene editing. At present, the transient expression vectors are the main usage in expressing Cas9 protein and sgRNA of cells, which may influence the cutting efficiency due to the low efficiency of this vector. And if a inducible expression cassette for Cas9 protein expression is inserted in the AAVS1 locus of human cells, the Cas9 protein expression can be expressed first. Then the cutting efficiency can be greatly improved through the transfection of small fragments sgRNA and the multiple sgRNA polygenic can also be edited at the same time. What's more, the inducible Cas9 protein expression can avoid the persistent expression of Cas9 proteins inside. As to the toxicity of cells, the persistent Cas9 protein expression can easily influence the experimental error. This paper plans to explore the insertion of inducible Cas9 protein expression cassette in AAVS1 locus of human cells by taking advantage of CRISPR/Cas9 technology.Firstly, aiming at AAVS1, this study constructs a plasmid LentiCRISPRv2-sgRNA 1/2/3 and finds the cutting efficiency is 22%,25% and 27% respectively. It also finds that the LentiCRISPRv2-sgRNA2/3 does not have off-target effect in its potential off-target locus.Secondly, by using the LentiCRISPRv2-sgRNA2/3 plasmid and AAV-CAGGS-EGFP donor plasmid, this study deals with the EGFP experiment of HEK293T and immortalized human tooth germ cells through the insertion of exogenous gene into AAVS1 locus. The results show that the LentiCRISPRv2-sgRNA2/3 plasmid and AAV-CAGGS-EGFP donor plasmid can insert EGFP expression element into the AAVS1 locus of these two cells and the insertion efficiency has no big difference.Finally, by using the LentiCRISPRv2-sgRNA2/3 plasmid and the inducible expression cassette for Cas9 protein expression, this study deals with the insertion of inducible Cas9 experiment into AAVS1 locus on HEK293T cells through Puro-Cas9 donor and Neo-M2rtTA donor plasmid. And the western blot results show that the inducible expression cassette for Cas9 can be inserted in human cells.