Characterization of CoCas9 nuclease from Capnocytophaga ochracea

RNA Biol. 2023 Jan;20(1):750-759. doi: 10.1080/15476286.2023.2256578. Epub 2023 Sep 24.

Abstract

Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5'-NRRWC-3' PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.

Keywords: CRISPR-Cas; Capnocytophaga ochracea; Cas9; CoCas9; RNA-guided nuclease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9* / genetics
  • CRISPR-Associated Protein 9* / metabolism
  • CRISPR-Cas Systems*
  • Capnocytophaga / genetics
  • Capnocytophaga / metabolism
  • Gene Editing
  • Humans

Substances

  • CRISPR-Associated Protein 9

Supplementary concepts

  • Capnocytophaga ochracea

Grants and funding

This work was supported by the Russian Science Foundation grant (21-14-00122). A. V. , P. S. and A. A. were supported by the Ministry of Science and Higher Education of the Russian Federation (project no. 075-15-2021-1062). M. K., T. A. were supported by the Ministry of Science and Higher Education of the Russian Federation under the program “Priority 2030”, 075-15-2021-1333, dated 30.09.2021. O. M. and K. S. were supported by the Russian Science Foundation grant (19-14-00323).