Research On Donor-adapted CRISPR/Cas9-Gal4BD Gene Editing System

The fast-rising CRISPR-derived gene editing technologies has been widely used in the researches of life science and biomedicine,plant and animal breeding.The emerging molecular design breeding based on the gene editing technologies make the rapid generation of animal germplasms possible.Inspired by the idea to drive the donor DNA to the double-stranded break(DSB),several modified CRISPR/Cas9 systems have been developed to improve HDR editing efficiency.Homologous recombination repair(HDR)is an important strategy for gene knock-in and precise editing,but its editing efficiency still needs to be improved.Inspired by the idea to drive the donor DNA to the double-stranded break(DSB),several modified CRISPR/Cas9 systems have been developed to improve HDR editing efficiency.In this paper,we propose the concept of Donor Adapting System(DAS)to summarize the gene editing strategies for modifying the CRISPR/Cas9 system with adaptors to drive the donor DNA fused to the adaptor.At the same time,a CRISPR/Cas9-Gal4 BD donor adaptation system was designed to fuse and express the DNA binding domain of the yeast Gal4 protein with the Cas9 protein.At the same time,PCR experiments were used to add specific binding sequence(BS)to the double-stranded DNA donor.Enabling the BS-ds DNA donor to bind to the Cas9-Gal4 BD fusion protein while recruiting at DBS to a higher effective concentration to improves HDR efficiency.The main results of this study are as follows:1.A total of 14 main expression vectors and related reporter vectors including CRISPR/Cas9-GGS1-Gal4 BD,CRISPR/Cas9-GGS3-Gal4 BD and CRISPR/Cas9-GGS5-G al4 BD were constructed by recombinant DNA technology.2.Introduce the DNA binding domain(Gal4 BD)of yeast transcription factor G al4 and the adaptor protein and adaptor sequence that can specifically recognize and bind the corresponding binding sequence(Binding Sequence,BS)into the CIRSPR/Ca s9 system and double-stranded DNA(ds DNA)donor.Construction of CIRSPR/Cas9-G al4 BD donor adaptation system.3.The CIRSPR/Cas9-Gal4 BD fusion method was further optimized at the level of the reporter vector,and the fusion design scheme using GGS5(5 repeated GGS sequences)as the linker and Gal4 BD protein fused to the C-terminus of the Cas9 protein was obtained.and express results with minimal impact.4.Further optimize the design of the BS-ds DNA donor at the level of the reporter vector,and obtain the result of higher HDR efficiency of the BS-ds DNA donor design scheme that adds a single BS sequence to the 5’ end of the PCR double-stranded donor.5.Precise editing of HEK293 T cell genome using the optimized Cas9-Gal4 BD fusion expression protocol and BS-ds DNA donor design protocol.The results after editing at four selected loci(AAVS1,EMX1.NUDT5,VEGFA)showed that the efficiency of HDR editing was improved at three loci.At the same time,it was found that the off-target effect of this system on the CRISPR/Cas9 system was reduced.