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GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos.
Nature Protocols ( IF 14.8 ) Pub Date : 2020-08-14 , DOI: 10.1038/s41596-020-0361-1
Erwei Zuo 1, 2 , Yidi Sun 1, 3 , Wu Wei 4, 5, 6 , Tanglong Yuan 2 , Wenqin Ying 1 , Hao Sun 7 , Liyun Yuan 4 , Lars M Steinmetz 6, 8, 9 , Yixue Li 4, 10, 11, 12, 13 , Hui Yang 1
Affiliation  

Genome editing holds great potential for correcting pathogenic mutations. We developed a method called 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. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq (https://github.com/sydaileen/GOTI-seq) for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.



中文翻译:

GOTI,一种识别小鼠胚胎基因组编辑的全基因组脱靶效应的方法。

基因组编辑具有纠正致病突变的巨大潜力。我们开发了一种称为 GOTI(双细胞胚胎注射全基因组脱靶分析)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。GOTI 直接比较编辑和未编辑的细胞,不受遗传背景的干扰,因此可以高灵敏度地检测潜在的脱靶变异。值得注意的是,GOTI 方法旨在通过结合实验和计算方法来检测任何基因组编辑工具的潜在脱靶变体,这对于准确评估基因组编辑工具的安全性至关重要。在这里,我们提供了 GOTI 的详细协议,包括小鼠交配、双细胞胚胎注射、胚胎第 14.5 天胚胎消化、荧光激活细胞分选、全基因组测序和数据分析。为了增强 GOTI 的实用性,我们还包括一个称为 GOTI-seq (https://github.com/sydaileen/GOTI-seq) 的计算工作流程,用于测序数据分析,它可以生成最终的全基因组脱靶变体直接来自原始测序数据。该协议通常需要 20 天从小鼠交配到测序,7 天用于测序数据分析。

更新日期:2020-08-14
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