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Title: Structural basis for Cas9 off-target activity.
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 or multiple non-canonical base pairs 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.  more » « less
Award ID(s):
1905374
NSF-PAR ID:
10351049
Author(s) / Creator(s):
Editor(s):
John Pham, Ph.D. Editor-in-Chief
Date Published:
Journal Name:
Cell
ISSN:
0092-8674
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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