Systematic Decomposition of Sequence Determinants Governing CRISPR/Cas9 Specificity

Authors

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

Publication Date

1-25-2022

Journal

Nature Communications

DOI

10.1038/s41467-022-28028-x

PMID

35078987

PMCID

PMC8789861

PubMedCentral® Posted Date

1-22-2022

PubMedCentral® Full Text Version

Post-print

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 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.

Keywords

Base Sequence, CRISPR-Cas Systems, Cell Line, Gene Editing, Humans, Mutation, Neoplasms, RNA, Guide, CRISPR-Cas Systems, CRISPR-Cas9 genome editing, Computational models, Biotechnology

Published Open-Access

yes

Recommended Citation

Fu, Rongjie; He, Wei; Dou, Jinzhuang; et al., "Systematic Decomposition of Sequence Determinants Governing CRISPR/Cas9 Specificity" (2022). Faculty, Staff and Student Publications. 2753.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/2753