Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells

Mol Cell. 2021 Sep 2;81(17):3637-3649.e5. doi: 10.1016/j.molcel.2021.07.035.


The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.

Keywords: CRISPR-Cas; CRISRP hybrid RNA-DNA; Cas9; chRDNA; off-target editing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Associated Protein 9 / physiology
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Associated Proteins / physiology
  • CRISPR-Cas Systems / genetics*
  • DNA / genetics
  • Endonucleases / genetics
  • Gene Editing / methods
  • Genetic Techniques
  • Genome / genetics
  • Genomics / methods
  • Humans
  • Leukocytes, Mononuclear / metabolism
  • Molecular Conformation
  • RNA, Guide, Kinetoplastida / genetics
  • Structure-Activity Relationship
  • T-Lymphocytes / metabolism*
  • T-Lymphocytes / physiology


  • CRISPR-Associated Proteins
  • RNA, Guide
  • DNA
  • CRISPR-Associated Protein 9
  • Endonucleases