Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos

First author: Erwei Zuo; Affiliations: Institute of Neuroscience, Chinese Academy of Sciences (中科院神经科学研究所): Shanghai, China

Corresponding author: Hui Yang

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.

基因组编辑使得纠正治病突变成为可能。但是，由于个体之间存在单碱基的多态性，所以很难检测基因组编辑的脱靶效应。本文，作者开发了一套叫做GOTI的方法来检测CRISPR-Cas9或是单碱基编辑技术编辑小鼠两细胞胚胎时期的一个卵裂球的脱靶效应。通过比较卵裂球编辑后和未编辑形成的子代细胞，作者发现由CRISPR-Cas9或是腺嘌呤碱基编辑技术编辑的胚胎中脱靶现象十分稀有，几乎与其自身的突变率相当。相反，胞嘧啶碱基编辑诱导的单核苷酸变异要高出20倍，说明该项技术可能需要进一步的改善以提高精度。

通讯：杨辉 (http://www.ion.ac.cn/chinese/laboratories/int.asp?id=96)

个人简介：2003-2007年，上海交通大学生命科学学院，生物技术学士；2007-2012年，中科院上海分院生化细胞所，发育生物学博士；2012-2013年，麻省理工Whitehead Institute，博士后。

研究方向：建立灵长类疾病模型。

doi: 10.1126/science.aav9973

Journal: Science

Published date: February 28, 2019