Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA

Nat Commun. 2020 Jul 17;11(1):3576. doi: 10.1038/s41467-020-17411-1.

Abstract

CRISPR/Cas9 is a programmable genome editing tool widely used for biological applications and engineered Cas9s have increased discrimination against off-target cleavage compared with wild-type Streptococcus pyogenes (SpCas9) in vivo. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. We show that DNA cleavage is impaired by more than 100- fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed rate of cleavage without increasing the rate of DNA rewinding and release. The kinetic partitioning favors release rather than cleavage of a bound off-target substrate only because the cleavage rate is so low. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.

Publication types

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

MeSH terms

  • CRISPR-Associated Protein 9 / chemistry
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA Cleavage
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism*
  • Kinetics
  • Streptococcus pyogenes / chemistry
  • Streptococcus pyogenes / enzymology*
  • Streptococcus pyogenes / genetics
  • Streptococcus pyogenes / metabolism

Substances

  • DNA, Bacterial
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes