Liang Zeyu, Wu Xin, Ye Zhaojin, She Lingwei, Ma Xiaoyan, Huo Yi-Xin
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China.
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China; Center for Future Foods, Muyuan Laboratory, 110 Shangding Road, Zhengzhou, Henan Province 450016, China; Beijing Institute of Technology (Tangshan) Translational Research Center, Hebei 063611, China.
STAR Protoc. 2025 Mar 21;6(2):103690. doi: 10.1016/j.xpro.2025.103690.
In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al..
在CRISPR-Cas9系统中，多个相同的单向导RNA（sgRNA）串联表达常常会引发同源序列丢失，这会影响多重基因组编辑效率。在此，我们展示了一种用于高通量筛选具有非重复突变体的功能性sgRNA的方案。我们描述了构建筛选平台、设计和构建sgRNA文库以及筛选sgRNA突变体的步骤。这些突变体可与Cas9蛋白相互作用，实现多重基因组编辑。有关此方案使用和执行的完整详细信息，请参阅Liang等人的文章。
