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. 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

SUBMITTER: Fu R 

PROVIDER: S-EPMC8789861 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature