Modification of alternative splicing in bovine somatic cell nuclear transfer embryos using engineered CRISPR-Cas13d

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.