数字农科院2.0

GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos

文献类型： 外文期刊

作者： 左二伟;;袁堂龙

作者机构：

期刊名称： Nature Protocols

ISSN： 1754-2189

年卷期： 2020 年 15 卷 9 期

页码：

收录情况： JCR（2021版）

摘要： 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.

分类号：

  • 相关文献
作者其他论文 更多>>