Animal cloning can be achieved by somatic cell nuclear transfer(SCNT),but the resulting live birth rate is relatively low.We previously improved the efficiency of bovine SCNT by exogenous melatonin treatment or by ove...Animal cloning can be achieved by somatic cell nuclear transfer(SCNT),but the resulting live birth rate is relatively low.We previously improved the efficiency of bovine SCNT by exogenous melatonin treatment or by overexpression of lysine-specific demethylase 4D(KDM4D)and 4E(KDM4E).In this study,we revealed abundant alternative splicing(AS)transitions during fertilization and embryonic genome activation,and demonstrated abnormal AS in bovine SCNT embryos compared with in vitro fertilized embryos.We used the CRISPR-Cas13d RNA-targeting system to target cis-elements of ABI2 and ZNF106 pre-m RNA to modify AS,thus reducing the ratio of abnormal-isoform SCNT embryos by nearly 50%and achieving a high survival rate(11%–19%).These results indicate that this system may provide an efficient method for bovine cloning,while also paving the way for further improvements in the efficiency of SCNT.展开更多
基金the National Natural Science Foundation of China(31802153,32072805 and 31872539)Special Funds for Talents in Northwest A&F University(Z111021512 and Z109021702)+1 种基金Innovation Project of Science and Technology in Shaanxi Province(2018JQ3035)National Major Project for Production of Transgenic Breeding(2016ZX08007-003)。
文摘Animal cloning can be achieved by somatic cell nuclear transfer(SCNT),but the resulting live birth rate is relatively low.We previously improved the efficiency of bovine SCNT by exogenous melatonin treatment or by overexpression of lysine-specific demethylase 4D(KDM4D)and 4E(KDM4E).In this study,we revealed abundant alternative splicing(AS)transitions during fertilization and embryonic genome activation,and demonstrated abnormal AS in bovine SCNT embryos compared with in vitro fertilized embryos.We used the CRISPR-Cas13d RNA-targeting system to target cis-elements of ABI2 and ZNF106 pre-m RNA to modify AS,thus reducing the ratio of abnormal-isoform SCNT embryos by nearly 50%and achieving a high survival rate(11%–19%).These results indicate that this system may provide an efficient method for bovine cloning,while also paving the way for further improvements in the efficiency of SCNT.