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Modification of alternative splicing in bovine somatic cell nuclear transfer embryos using engineered CRISPR-Cas13d.
Science China Life Sciences Pub Date : 2022-05-05 , DOI: 10.1007/s11427-021-2060-x
Rui Cheng 1 , Xiaoman Zheng 1 , Yingmei Wang 1 , Xing Ma 1 , Xin Liu 1 , Wenjun Xu 1 , Mengyun Wang 1 , Yuanpeng Gao 1 , Xupeng Xing 1 , Chuan Zhou 1 , Hongzheng Sun 1 , Zekun Guo 1 , Fusheng Quan 1 , Jun Liu 1 , Song Hua 1 , Yongsheng Wang 1 , Yong Zhang 1 , Xu Liu 1
Affiliation  

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.

中文翻译:

使用工程化 CRISPR-Cas13d 修饰牛体细胞核移植胚胎中的选择性剪接。

动物克隆可以通过体细胞核移植(SCNT)实现,但由此产生的活产率相对较低。我们以前通过外源性褪黑激素处理或赖氨酸特异性脱甲基酶 4D (KDM4D) 和 4E (KDM4E) 的过表达提高了牛 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选择性剪接 (AS) 转换,并证明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靶向系统靶向ABI2和ZNF106 pre-mRNA的顺式元件来修饰AS,从而将异常异构体SCNT胚胎的比例降低近50%,并实现高存活率(11%- 19%)。这些结果表明该系统可能为牛的克隆提供一种有效的方法,
更新日期:2022-05-05
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