Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Center for Animal Genomics, Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518124, China.
Science. 2019 Apr 19;364(6437):289-292. doi: 10.1126/science.aav9973. Epub 2019 Feb 28.
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 and nonedited blastomeres at embryonic day 14.5 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. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.
基因组编辑有望纠正致病突变。然而，由于个体中单核苷酸多态性的存在，编辑的脱靶效应很难确定。在这里，我们开发了一种名为 GOTI（通过两细胞胚胎注射进行全基因组脱靶分析）的方法，用于通过使用 CRISPR-Cas9 或碱基编辑器编辑两细胞小鼠胚胎的一个卵裂球来检测脱靶突变。在胚胎第 14.5 天比较编辑和未编辑卵裂球的后代细胞的全基因组序列表明，CRISPR-Cas9 或腺嘌呤碱基编辑器编辑的胚胎中脱靶单核苷酸变体 (SNV) 很少，频率接近自发突变率。相比之下，胞嘧啶碱基编辑以超过 20 倍的更高频率诱导 SNV，需要解决其保真度的问题。
