Closely related type II-C Cas9 orthologs recognize diverse PAMs

Elife. 2022 Aug 12:11:e77825. doi: 10.7554/eLife.77825.

Abstract

The RNA-guided CRISPR/Cas9 system is a powerful tool for genome editing, but its targeting scope is limited by the protospacer-adjacent motif (PAM). To expand the target scope, it is crucial to develop a CRISPR toolbox capable of recognizing multiple PAMs. Here, using a GFP-activation assay, we tested the activities of 29 type II-C orthologs closely related to Nme1Cas9, 25 of which are active in human cells. These orthologs recognize diverse PAMs with variable length and nucleotide preference, including purine-rich, pyrimidine-rich, and mixed purine and pyrimidine PAMs. We characterized in depth the activity and specificity of Nsp2Cas9. We also generated a chimeric Cas9 nuclease that recognizes a simple N4C PAM, representing the most relaxed PAM preference for compact Cas9s to date. These Cas9 nucleases significantly enhance our ability to perform allele-specific genome editing.

Keywords: CRISPR/Cas9; Nme1Cas9 orthologs; Nsp2Cas9; PAM diversity; crispr/cas9; genetics; genome editing; genomics; nme1cas9 orthologs; nsp2cas9; pam diversity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9* / metabolism
  • CRISPR-Cas Systems*
  • Endonucleases / genetics
  • Gene Editing
  • Humans
  • Purines
  • Pyrimidines

Substances

  • Purines
  • Pyrimidines
  • CRISPR-Associated Protein 9
  • Endonucleases

Grants and funding

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.