Abstract
The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of non-canonical base pairing interactions and preservation of base stacking within the guide–off-target heteroduplex. Off-target sites containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping rather than RNA bulge formation. Additionally, PAM-distal mismatches result in duplex unpairing and induce a conformational change of the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.
Competing Interest Statement
P.D.D. and M.J.I are current employees of Caribou Biosciences, Inc., and C-H.L. and P.C. are former employees of Caribou Biosciences, Inc. M.J. is a co-founder of Caribou Biosciences, Inc. M.J., C-H.L., M.J.I., P.C. and P.D.D. are named inventors on patents and patent applications related to CRISPR-Cas technologies.
