A High Efficient CRISPR/Cas9-based Biallelic Genome Editing System And Its Application In Pig Genome Editing

Genome editing is a new technology that can be used to precisely make a deletion,mutation or insertion within a given genome target locus,which leverages the machanims of the cellular DNA double strand break(DSB)repair to generate InDels or enhance the homology directed recombination(HDR).The effiency of tradition gene targeting is extremely low due to the inefficient spontaneous homologous recombination within mammlian cells.Since its advent,the custom-designed nucleases has revulionized the research field of genome engineering.The mechanism of high efficient genome editing is based on the discovery that the DNA double strand break(DSB)significantly enhances the efficiency of precisely genome editing contributed by cellular DNA repair system.With the more genome sequence databases avalable,the focus of post genome era is the gene function study.Genome editing technology is an important and powerful tool in elucidating gene functions.CRISPR/Cas9 has emerged as the newest genome editing technology that has been widely used in genome editing study of varieties of organisms due to its simple,reliable and high efficient features.CRISPR/Cas9 system has potential applications not only in gene fuction study,but also in developing human disease models,gene therapy as well as generation of transgenic animals.Screening and enriching biallelic genome modified cells are often time-consuming,low efficiency and technique challenging.In this research,a surrogate reporter system coupled with a knocked-in donor system(Rep/Don)was developed for a novel high efficient biallelic CRISPR/Cas9 gene editing.The Rep/Don system can not only serve as homologous repair donors but also at the same time function as surrogate reporter used for secreening the functional Cas9 paired with sgRNAs of high targeting efficiency.At first,the Rep/Don knocked-in system was tested at the protein-coding gene loci in mammalian cells,which resulted in high biallelic knocked-in efficiency.The next,the system was used in pig PK15 cells for targeting a long non-coding RNA gene loci,and also had higher efficiency in receiving biallelic genome modified cells.1.The Rep/Don PG,Rep/Don NR and Rep/Don ZR knocked-in plasmids andcorresponding CRISPR/Cas9 expression plasmids were constructed for mammalian cell genome editing.Two surrogate reporter cassettes(Puror-eGFP and Zeor-mRFP)were incorporated into paired donor plasmids.The antibiotic resistant gene was interrupted by a sgRNA target sequence flanked by a direct repeats(~200 bp in length)as SSA arms.The fluorescent protein gene was fused after Puror gene by T2 A with the incorrect ORF,which were used as both the surrogate reporters and the knock-in donors.If the designed CRISPR/Cas9 nuclease could work,double strand breaks(DSBs)would be generated on the target sequence within the surrogate reporter cassettes of the donor plasmids.The DSBs then trigger the repair of the surrogate reporter cassettes,leading to functional expression of the reporter genes.At the same time,the CRISPR/Cas9 would also generate DSBs on the genomic target sequences,triggering the homology directed repair(HDR)based integration of the previously repaired surrogate reporter cassettes,with the reporter genes further applied as selecting marker genes.2.We demonstrated the high efficiency of our dual-surrogate reporter-integrated donor system for genome targeting in human HEK293 T cells,with the biallelic frequency of 2.33%,2.08% and 34.09% in groups Rep/Don PG,Rep/Don ZR and Rep/Don PG+ZR respectively.3.The best concentration of Puromycin,G418 and Zeocin were optimized as 1 ?g/mL,1200 ?g/mL and 400 ?g/m L respectively,according to the drug killing assay.4.Three sgRNA were designed for pig lnc-sscg3623 gene,and the efficiency of these sgRNAs were detected,the results showed the higher activity in Tar 2 and Tar 1.Then,Rep/Don PG,Rep/Don NR and Rep/Don ZR knoncked-in systems and CRISPR/Cas9 targeting plasmids were construced for lnc-sscg3623 gene Tar 2 and Tar 1 loci respectively.5.The high efficiency of our dual-surrogate reporter-integrated donor system was demonstrated for genome targeting in pig PK15 cells.At Tar 1 locus,the monoallelic frequency was 25%,37.5% and 26.32% in goups Rep/Don PG,Rep/Don NR and Rep/Don ZR respectively,and the biallelic frequency of group Rep/Don PG+ZR was 10%.On the Tar 2site,with the monoallelic frequency of 75%,65% and 64.86%,and the biallelic frequency of6.81%,7.5% and 2.7% in groups Rep/Don PG,Rep/Don NR and Rep/Don ZR respectively.The biallelic frequency of paired groups Rep/Don PG+NR and Rep/Don PG+ZR were16.67% and 18.18% respectively.This study provide a powerful genetic tool for assisting the selection and enrichment of cells with targeted biallelic genome modification,which could provide foundation for studying gene function and improvement of economic traits in livestock breeding.