Electroporation Transfection Of Cas9 RNP To Edit ACTG1 Gene In Sheep Zygotes

Objective: Based on the in vitro enzyme cleavage and sheep fibroblast transfection experiments,specifically targeting the Fec B SNP locus,a major functional gene associated with high reproductive capacity in sheep,and the termination codon site of the ACTG1 gene encoding γ-actin(Actin Gamma 1),this research aims to determine the activity of the purified Cas9 RNPs and explore the potential application of Cas9 RNPs for sheep Fec B gene genotyping.Methods: Cas9 protein and transcribed sg RNA were co-expressed in E.coli induced by IPTG.Then Cas9 RNPs were purified by Ni-NTA arose medium and concentrated by ultrafiltration tube.The sheep with the homozygous type(BB),heterozygous type(B+),and wild type(++)of Fec B gene were used as materials,and specific primers of target sites of Fec B gene were amplified as substrates.Purified Cas9 RNPs were used to perform enzyme digestion detection on target samples of different Fec B genotypes.The primary cultured sheep fibroblasts were obtained by tissue mass culture and electrotransfected with Cas9 RNPs into sheep fibroblasts,the cells were collected,and the genomes were extracted for PCR amplification to detect the effect of genome editing.The Cas9 RNPs of the codon target was terminated by ACTG1,and the Cas9 RNPs was delivered by electroperforation and imported into the sheep parthenogenetic activated embryos.The morula and blastula development rates were measured at different perforation voltages for 7 days in vitro,and the genome was extracted for PCR amplification to detect the genome mutation rate.Results: 1: It is an effective method to co-express Cas9 and sg RNA in E.coli and purify Cas9 RNPs with good stability and in vitro nicking activity.2: Sheep fibroblasts were electrotransfected with Cas9 RNPs,and the mutation detection results showed that the purified Cas9 RNPs had good genome editing activity.The appropriate voltage was determined by electroporation of sheep parthenogenetic activated embryos according to blastocyst development rate.Conclusion: Co-expression of Cas9 RNPs in E.coli is an effective method for low-cost preparation.After optimizing the perforation voltage,the suitable voltage for electroperforation of sheep parthenogenetic activated embryos was 25 V or 30 V.