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
Harlequin ichthyosis (HI) is a severe genetic skin disorder and caused by mutation in the ATP-binding cassette A12 (ABCA12) gene. The retinoid administration has dramatically improved long-term survival of HI, but imp...Harlequin ichthyosis (HI) is a severe genetic skin disorder and caused by mutation in the ATP-binding cassette A12 (ABCA12) gene. The retinoid administration has dramatically improved long-term survival of HI, but improvements are still needed. However, the ABCA12 null mice failed to respond to retinoid treatment, which impedes the development of novel cure strategies for HI. Here we generated an ethylnitrosourea mutagenic HI pig model (named Z9), which carries a novel deep intronic mutation IVS49-727 A>G in the ABCA12 gene, resulting in abnormal mRNA splicing and truncated protein production. Z9 pigs exhibit significant clinical symptom as human patients with HI. Most importantly, systemic retinoid treatment significantly prolonged the life span of the mutant pigs via improving epidermal maturation, decreasing epidermal apoptosis, and triggering the expression of ABCA6. Taken together, this pig model perfectly resembles the clinical symptom and molecular pathology of patients with HI and will be useful for understanding mechanistic insight and developing therapeutic strategies.展开更多
Pigs are one of the most important domesticated animals and have great value in agriculture and biomedicine.Single nucleotide polymorphisms(SNPs)are a dominant type of genetic variation among individual pigs and contr...Pigs are one of the most important domesticated animals and have great value in agriculture and biomedicine.Single nucleotide polymorphisms(SNPs)are a dominant type of genetic variation among individual pigs and contribute to the formation of traits.Precision single base substitution provides a strategy for accurate genetic improvement in pig production with the characterization of functional SNPs and genetic variants in pigs.Base editing has recently been developed as the latest gene-editing tool that can directly make changes in single nucleotides without introducing double-stranded DNA breaks(DSBs),providing a promising solution for precise genetic modification in large animals.This review summarizes gene-editing developments and highlights recent genetic dissection related to SNPs in major economic traits which may have the potential to be modified using SNP-editing applications.In addition,limitations and future directions of base editing in pig breeding are discussed.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.
基金supported by the National Key Research and Development Program of China (2020YFC1316602, 2016YFC0905100, and 2020YFA0509503)the National Natural Science Foundation of China (31925036, 81788101, 81671274, 31801031, 81230015, and 31701073)+3 种基金the Strategic Priority Research Programs of CAS (XDA16030101)the National Transgenic Project of China (2016ZX08009003-006-007)the CAMS Innovation Fund for Medical Sciences (2016-I2M-1002)the Central Research Institutes of Basic Research and Public Service Special Operations (2017PT31016)。
基金the Strategic Priority Research Programs of CAS(XDA16030300)the National Natural Science Foundation of China(81671274,31272440,and 31801031)+1 种基金the National Transgenic Project of China(2016ZX08009003-006-007)the Elite Youth Program of the Chinese Academy of Agricultural Sciences(ASTIP-IAS05).
文摘Harlequin ichthyosis (HI) is a severe genetic skin disorder and caused by mutation in the ATP-binding cassette A12 (ABCA12) gene. The retinoid administration has dramatically improved long-term survival of HI, but improvements are still needed. However, the ABCA12 null mice failed to respond to retinoid treatment, which impedes the development of novel cure strategies for HI. Here we generated an ethylnitrosourea mutagenic HI pig model (named Z9), which carries a novel deep intronic mutation IVS49-727 A>G in the ABCA12 gene, resulting in abnormal mRNA splicing and truncated protein production. Z9 pigs exhibit significant clinical symptom as human patients with HI. Most importantly, systemic retinoid treatment significantly prolonged the life span of the mutant pigs via improving epidermal maturation, decreasing epidermal apoptosis, and triggering the expression of ABCA6. Taken together, this pig model perfectly resembles the clinical symptom and molecular pathology of patients with HI and will be useful for understanding mechanistic insight and developing therapeutic strategies.
基金supported by the National Natural Science Foundation of China(81671274,31925036,31272440,and 31801031)the National Transgenic Project of China(2016ZX08009003006-007)the Elite Youth Program of the Chinese Academy of Agricultural Sciences(ASTIP-IAS05)。
文摘Pigs are one of the most important domesticated animals and have great value in agriculture and biomedicine.Single nucleotide polymorphisms(SNPs)are a dominant type of genetic variation among individual pigs and contribute to the formation of traits.Precision single base substitution provides a strategy for accurate genetic improvement in pig production with the characterization of functional SNPs and genetic variants in pigs.Base editing has recently been developed as the latest gene-editing tool that can directly make changes in single nucleotides without introducing double-stranded DNA breaks(DSBs),providing a promising solution for precise genetic modification in large animals.This review summarizes gene-editing developments and highlights recent genetic dissection related to SNPs in major economic traits which may have the potential to be modified using SNP-editing applications.In addition,limitations and future directions of base editing in pig breeding are discussed.
