Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos
Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos
复制标题
胞嘧啶碱基编辑器在小鼠胚胎中产生大量脱靶单核苷酸变异
DOI：
10.1126/science.aav9973
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发表时间：
2019-04-19
期刊：
影响因子：
56.9
通讯作者：
Yang, Hui
中科院分区：
文献类型：
--
作者：
Zuo, Erwei;Sun, Yidi;Yang, Hui
非预期的基因组修饰限制了基因编辑工具的潜在治疗用途。发现脱靶的可用方法通常在体内不起作用或检测单核苷酸变化。本期的三篇论文报告了体内监测基因编辑工具的新方法（见Kempton和Qi的观点）。Wienert等人跟踪了DNA修复蛋白对CRISPR-Cas9诱导的DNA断裂的募集，从而能够在细胞和动物模型中无偏地检测脱靶编辑。Zuo等人通过将碱基编辑器注射到双细胞小鼠胚胎的一个卵裂球中，并使另一个遗传上相同的卵裂球未经编辑，在没有天然遗传异质性干扰的情况下鉴定了脱靶。Jin等人对单个基因组编辑的水稻植物进行了全基因组测序，以识别意外突变。胞嘧啶，但不是腺嘌呤，碱基编辑器在小鼠和水稻中诱导了许多单核苷酸变体。《科学》，本期第286页，第289页，第292页;另见第234页，小鼠胚胎中的胞嘧啶碱基编辑产生了许多脱靶的单核苷酸变体。基因组编辑有望纠正致病性突变。然而，由于个体中的单核苷酸多态性，很难确定编辑的脱靶效应。在这里，我们开发了一种名为GOTI（通过双细胞胚胎注射进行全基因组脱靶分析）的方法，通过使用CRISPR-Cas9或碱基编辑器编辑双细胞小鼠胚胎的一个卵裂球来检测脱靶突变。对胚胎第14.5天编辑和未编辑卵裂球后代细胞的全基因组序列进行比较，结果显示，在CRISPR-Cas9或腺嘌呤碱基编辑器编辑的胚胎中，脱靶单核苷酸变异（SNV）非常罕见，频率接近自发突变率。相比之下，胞嘧啶碱基编辑以超过20倍的频率诱导SNV，需要解决其保真度的解决方案。
Spotting off-targets from gene editing Unintended genomic modifications limit the potential therapeutic use of gene-editing tools. Available methods to find off-targets generally do not work in vivo or detect single-nucleotide changes. Three papers in this issue report new methods for monitoring gene-editing tools in vivo (see the Perspective by Kempton and Qi). Wienert et al. followed the recruitment of a DNA repair protein to DNA breaks induced by CRISPR-Cas9, enabling unbiased detection of off-target editing in cellular and animal models. Zuo et al. identified off-targets without the interference of natural genetic heterogeneity by injecting base editors into one blastomere of a two-cell mouse embryo and leaving the other genetically identical blastomere unedited. Jin et al. performed whole-genome sequencing on individual, genome-edited rice plants to identify unintended mutations. Cytosine, but not adenine, base editors induced numerous single-nucleotide variants in both mouse and rice. Science, this issue p. 286, p. 289, p. 292; see also p. 234 Cytosine base editing in mouse embryos generates numerous off-target single-nucleotide variants. 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.