Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity
Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity
Abstract The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. Here we systematically investigated the sequence determinants governing CRISPR/Cas9 specificity by measuring the off-on ratios of 1,902 gRNAs on 13,314 target sequences using an improved synthetic system with dual-target design. Our study revealed a comprehensive set of rules including 3 factors in CRISPR/Cas9 off-targeting: 1) the nucleotide context and position of a single mismatch; 2) an “epistasis-like” combinatorial effect of multiple mismatches; and 3) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context. Notably, the combinatorial effect and GMT are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. Based on these rules, we developed a model-based off-target prediction tool (MOFF), which showed superior performance compared to the existing methods.
He Wei、Dou Jinzhuang、Hou Connie、Wang Yalong、Gao Xue Sherry、Chen Yiwen、Wang Helen、Bedford Ella、Depken Martin、Xu Han、Villarreal Oscar D.、Zhang Liang、Fu Rongjie
Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDepartment of Chemical and Biomolecular Engineering, Rice UniversityDepartment of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterKavli Institute of NanoScience and Department of BionanoScience, Delft University of TechnologyDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center||Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center||The Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer CenterDepartment of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center
生物科学研究方法、生物科学研究技术分子生物学基础医学
He Wei,Dou Jinzhuang,Hou Connie,Wang Yalong,Gao Xue Sherry,Chen Yiwen,Wang Helen,Bedford Ella,Depken Martin,Xu Han,Villarreal Oscar D.,Zhang Liang,Fu Rongjie.Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity[EB/OL].(2025-03-28)[2025-06-24].https://www.biorxiv.org/content/10.1101/2021.08.02.454843.点此复制
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