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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing

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Structural basis for Cas9 off-target activity

Martin Pacesa1, Chun-Han Lin2, Antoine Cléry3

  • 1Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Cell
|October 28, 2022
PubMed

Insights

CRISPR-Cas9 genome editing can target unintended DNA sequences, raising safety concerns. Structural analysis reveals noncanonical base pairing and accommodation of deletions enable off-target binding, guiding improved guide RNA design.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • CRISPR-associated (Cas) nuclease Cas9 is a powerful genome editing tool.
  • Cas9 specificity relies on guide RNA complementarity to target DNA.
  • Off-target cleavage by Cas9 poses safety risks for clinical applications.

Purpose of the Study:

  • To elucidate the structural basis of Cas9 off-target binding and cleavage.
  • To understand how Cas9 accommodates mismatches and deletions in target DNA.
  • To inform the rational design of safer Cas9-based genome editing systems.

Main Methods:

  • X-ray crystallography of Cas9-DNA complexes.
  • Analysis of off-target substrates with varying complementarity.
  • Structural comparison of on-target and off-target binding modes.

Main Results:

  • Cas9 binds off-target DNA via noncanonical base-pairing interactions.
  • Single-nucleotide deletions are accommodated by base skipping or multiple noncanonical pairs.
  • PAM-distal mismatches induce duplex unpairing and conformational changes in Cas9.

Conclusions:

  • Structural insights explain Cas9 off-target activity.
  • Findings facilitate improved guide RNA design for enhanced specificity.
  • This work aids in developing better off-target prediction algorithms for CRISPR technology.
Keywords:
CRISPRCas9X-ray crystallographybase pairinggenome editingguide RNAmismatchnucleaseoff-target

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