College of Veterinary Medicine, Northwest A&F University, Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Yangling, 712100, China.
Sci China Life Sci. 2022 Nov;65(11):2257-2268. doi: 10.1007/s11427-021-2060-x. Epub 2022 May 5.
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.
动物克隆可以通过体细胞核移植（SCNT）来实现，但由此产生的活产率相对较低。我们之前通过外源性褪黑素处理或过表达赖氨酸特异性去甲基酶 4D（KDM4D）和 4E（KDM4E）来提高牛 SCNT 的效率。在这项研究中，我们揭示了受精和胚胎基因组激活过程中丰富的可变剪接（AS）转换，并证明了与体外受精胚胎相比，牛 SCNT 胚胎中的 AS 异常。我们使用 CRISPR-Cas13d RNA 靶向系统来靶向 ABI2 和 ZNF106 前体 mRNA 的顺式元件以修饰 AS，从而将异常同种型 SCNT 胚胎的比例降低近 50%，并实现了高存活率（11%-19%）。这些结果表明，该系统可为牛克隆提供一种有效方法，同时也为进一步提高 SCNT 的效率铺平了道路。
