Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity

Rongjie Fu, Wei He, Jinzhuang Dou, Oscar D. Villarreal, Ella Bedford, Helen Wang, Connie Hou, Liang Zhang, Martin Depken, More Authors

Research output: Contribution to journalArticleScientificpeer-review

4 Citations (Scopus)
15 Downloads (Pure)


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. To systematically explore the sequence determinants governing CRISPR/Cas9 specificity, here we describe a dual-target system to measure the relative cleavage rate between off- and on-target sequences (off-on ratios) of 1902 gRNAs on 13,314 synthetic target sequences, and reveal a set of sequence rules involving 2 factors in off-targeting: 1) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context; 2) an “epistasis-like” combinatorial effect of multiple mismatches, which are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. These sequence rules lead to the development of MOFF, a model-based predictor of Cas9-mediated off-target effects. Moreover, the “epistasis-like” combinatorial effect suggests a strategy of allele-specific genome editing using mismatched guides. With the aid of MOFF prediction, this strategy significantly improves the selectivity and expands the application domain of Cas9-based allele-specific editing, as tested in a high-throughput allele-editing screen on 18 cancer hotspot mutations.

Original languageEnglish
Article number474
Number of pages15
JournalNature Communications
Issue number1
Publication statusPublished - 2022


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