Gal4BD-Cas9 Improved Gene Targeting And Recombiantion In Mammalian Cells

Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location. The ability of precise gene editing is essential for many experimental paradigms such as functional analysis and gene therapy applications. DNA breaks made by the clustered,regularly interspaced, short palindromic repeats(CRISPR)/cas9 system from Streptococcus pyogenes stimulate repair parthway either NHEJ or HR. Precise gene editing is a genetic technique to insert or deletion sepecific DNA sequence in its genomic location via homologous recombination(HR). But the low efficiency of HR often limits the application of gene targeting. Therefore, it is of great significance to improve the efficiency of homologous recombination repair for gene targeting modification.Here, we exploit a novel approach to increase the HR efficiency, in which we bind a site specific DNA binding protein-Gal4 BD by fusion expression with CRISPR-Cas9 to a donor molecule target a specific genetic locus for correction. Functional repair donor containing a sequence that can bind to Gal4 BD were designed as part of a longer DNA molecule that contained a region with homology to repair an CRISPR/Cas9 generated double-strand break and correct the disrupted GFP gene. For double strand break(DSB) in genome DNA can induce gene repair by either NHEJ or HR, We hypothesis that we can use Gal4BD-Cas9 to creat a double strand break in target site. There will be Green fluorescence when PCR donnors repair the GFP gene. We want to increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells by this concept.In our research, first we construct a reporter cell line which disrupted GFP integration in HEK-293 T genome by Piggybac transposons. After puromycine selection for 10 days, we pick out positive monoclonal cells and extracted cell genome to detecte the reporter gene integration. Next, we constructed the Gal4BD-Cas9 systerm including Gal4BD-Cas9 fusion expression vector and different length HR donor amplified by PCR, which contain UAS sequnce and homology sequence with GFP. After cotransfection with fusion expression vector and PCR donor, the number of green fluorescent cells was calculated, and the efficiency of homologous recombination repair was calculated.Our experiment shows that the Gal4 BD binding sequence PCR donor stimulated precise gene editing up to 4 folds in human 293 T cells in which we integrate disrupted GFP reporter when the PCR donor contain a Homology arm as long as 60 bp. Our new approach provides useful tools and a new concept to improve the frequency of precise gene modifications in mammalian cells.