Study On Single Base Mutation Of Functional Domains Of Porcine MSTN And CD163 Genes

There are 83 indigenous pig breeds in China,which have the advantages of high reproductive rate and high intramuscular fat content.They are unique genetic resources and valuable materials for germplasm innovation and utilization.However,the disadvantages of low lean meat rate and weak disease resistance hinder their popularization and application.Myostatin(MSTN)is a major gene regulating skeletal muscle growth and development,and deletion or knockout of this gene can increase the lean meat percentage and lower the fat content of the animals.The scavenger receptor CD163 is one of the key receptors for porcine reproductive and respiratory syndrome virus PRRSV infection of host cells.Porcine CD163SRCR5 is a key region that binds to PRRSV,and knocking out this region could block PRRSV virus infection in pigs.Compared with the traditional CRISPR/Cas9 gene editing technology,the single-base editing technology can mutate a single base directionally and does not rely on DNA double-strand breaks,which not only avoids the randomness of nonhomologous end joining repair pathway,but also gets rid of limitation of the inefficiency of homologous recombination,and it is now widely used in species improvement.Therefore,in this study,pig lean meat rate gene MSTN and the disease resistance gene CD163 were used as key sites,stop codons were introduced to the second exon of MSTN and the fifth exon of CD163 to prepare gene knockout of monoclonal cell lines,laying a foundation for later breeding of single-base editing pigs.The main contents of this study are as follows:(1)The primary fibroblasts from Ningxiang pig kidney were successfully obtained.The cells were fibrous and angular,with good viability,which proved that the cells were isolated successfully.(2)Using the single guide RNA(single guide RNA,sg RNA)design tool,the sg RNAs of the two genes were designed respectively,and the recombinant expression vectors p MLM3636-puro-MSTN/p MLM3636-puro-CD163 targeting MSTN and CD163 genes were successfully constructed through the steps of annealing,plasmid ligation,transformation,bacterial picking and propagation.(3)In order to verify the editing efficiency of BE4max-P2A-GFP and YE1-BE3-FNLS,they were transfected into HEK 293 T cells with lipofection,respectively.When the cell number was 2 × 105,the plasmid transfection quantity was 8 μg and Lipofectamine3000 volume was 7.5 μL,the transfection efficiency was highest,and has minimal effect on cell viability.The sequencing results showed that both editors were able to edit the target site.The editing efficiency of sg RNAs with different sequences was different,but the editing efficiency of YE1-BE3-FNLS was generally higher than that of BE4max-P2A-GFP.(4)To explore the editing efficiency of BE4max-P2A-GFP and YE1-BE3-FNLS in kidney fibroblasts,BE4max-P2A-GFP or YE1-BE3-FNLS and p MLM3636-puroMSTN/p MLM3636-puro-CD163 were co-transferred into kidney fibroblasts by lipofection and electrotransfection,respectively.The results showed that the transfection efficiency of electrotransfection was higher than that of lipofection.The editing efficiency of YE1-BE3-FNLS at C3 was 35% higher than that of BE4max-P2A-GFP.(5)Electrotransfection method was used to transfect kidney fibroblasts,and positive monoclonal cell lines were obtained by red fluorescence and puromycin screening.Using YE1-BE3-FNLS and MLM3636-puro-MSTN to edit MSTN gene,55 monoclonal cell lines were obtained,of which 3 cell lines(10#,25#,51#)had C→T mutation at the target site,and9 cell lines were in C→T mutation occurred at the non-target site,and the target mutation rate was(3/55)5.5%.Meanwhile,40 monoclonal cell lines were obtained by editing the CD163 gene with YE1-BE3-FNLS and MLM3636-puro-CD163,of which 2 cell lines(14#,25#)had directional C→T mutation at the target site,the target mutation rate was(2/40)5.0%,and 1cell line had non-directional C→G mutation at the target site.(6)Fluorescence quantification and Western blotting analysis were performed on the mutant positive cells.The results of fluorescence quantification showed that there was no significant difference in the expression levels of the control group and the mutant group in the transcriptome level.Western blotting results showed that,compared with the wild group,the protein expression of MSTN in cell line 10# decreased by about 60%,and the protein expression of CD163 in cell line 25# decreased by about 20%.Conclusion: The editing efficiency of YE1-BE3-FNLS was higher than that of BE4maxP2A-GFP;the YE1-BE3-FNLS single-base editor successfully introduced stop codons in the coding regions of MSTN and CD163 advancely to terminate their translation,and their protein expression were significantly decreased.A single-base editing system for gene knockout in kidney fibroblasts was established,laying a foundation for breeding MSTN and CD163 base-editing pigs.