Structural basis for Cas9 off-target activity

Cell. 2022 Oct 27;185(22):4067-4081.e21. doi: 10.1016/j.cell.2022.09.026.


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 noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in 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.

Keywords: CRISPR; Cas9; X-ray crystallography; base pairing; genome editing; guide RNA; mismatch; nuclease; off-target.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Pairing
  • CRISPR-Cas Systems*
  • Endonucleases / metabolism
  • Gene Editing
  • Nucleotides
  • RNA, Guide, Kinetoplastida* / metabolism


  • RNA, Guide
  • Endonucleases
  • Nucleotides