Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity

Rongjie Fu; Wei He; Jinzhuang Dou; Oscar David Villarreal; Ella Bedford; Helen H. Wang; Connie Hou; Liang Zhang; Yalong Wang; Dacheng Ma; Yiwen Chen; Xue Gao; Martin Depken; Han Xu

doi:10.1038/s41467-022-28028-x

Systems BiologyNature CommunicationsJan 25, 2022

Peer-reviewed journal article

Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity

Rongjie Fu, Wei He iD, Jinzhuang Dou iD, Oscar David Villarreal iD, Ella Bedford, Helen H. Wang iD, Connie Hou iD, Liang Zhang iD, Yalong Wang iD, Dacheng Ma iD, Yiwen Chen iD, Xue Gao iD, Martin Depken iD, Han Xu iD

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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.