The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors(CBEs) that convert C:G to T:A have emerged as p...The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors(CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations.Our process resulted in a stop codon in CD163 and myostatin(MSTN) and introduced a beneficial allele in insulin-like growth factor-2(IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection,providing a potential new route to produce elite pig breeders.展开更多
基金supported by the National Natural Science Foundation for Distinguished Young Scholars (31925036)the National Key Research and Development Program of China (2020YFC1316602)+2 种基金the National Natural Science Foundation of China (81671274, 31272440, and 31801031)the National Transgenic Project of China (2016ZX08009003-006-007)the Elite Youth Program of the Chinese Academy of Agricultural Sciences (ASTIP-IAS05)
文摘The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors(CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations.Our process resulted in a stop codon in CD163 and myostatin(MSTN) and introduced a beneficial allele in insulin-like growth factor-2(IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection,providing a potential new route to produce elite pig breeders.
