Abstract
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-mRNA 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.
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Data and materials availability
All data needed to evaluate the conclusions in the paper are presented in the paper and/or the Supplementary Materials. The RNA-sequencing data used in this study were obtained from the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE38495, GSE45719 and GSE121227.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (31802153, 32072805 and 31872539), Special Funds for Talents in Northwest A&F University (Z111021512 and Z109021702), Innovation Project of Science and Technology in Shaanxi Province (2018JQ3035) and National Major Project for Production of Transgenic Breeding (2016ZX08007-003). We thank Susan Furness, PhD, from Liwen Bianji (https://www.liwenbianji.cn/) for editing the English text of a draft of this manuscript.
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Compliance and ethics All animal experiments were performed in strict accordance with the Guidelines for the Care and Use of Animals of Northwest A&F University. All experiments were approved by the Care and Use of Animals Centre, Northwest A&F University. All precautions were performed to minimize animal suffering.
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Cheng, R., Zheng, X., Wang, Y. et al. Modification of alternative splicing in bovine somatic cell nuclear transfer embryos using engineered CRISPR-Cas13d. Sci. China Life Sci. 65, 2257–2268 (2022). https://doi.org/10.1007/s11427-021-2060-x
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DOI: https://doi.org/10.1007/s11427-021-2060-x