TY - JOUR
T1 - Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity
AU - Fu, Rongjie
AU - He, Wei
AU - Dou, Jinzhuang
AU - Villarreal, Oscar D.
AU - Bedford, Ella
AU - Wang, Helen
AU - Hou, Connie
AU - Zhang, Liang
AU - Depken, Martin
AU - More Authors, null
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85123782732&partnerID=8YFLogxK
U2 - 10.1038/s41467-022-28028-x
DO - 10.1038/s41467-022-28028-x
M3 - Article
C2 - 35078987
AN - SCOPUS:85123782732
SN - 2041-1723
VL - 13
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 474
ER -