Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii

Int J Mol Sci. 2022 Aug 18;23(16):9289. doi: 10.3390/ijms23169289.

Abstract

Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from Ruminococcus bromii sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5'-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to Acidaminococcus sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing.

Keywords: CRISPR; Cas endonuclease; genome editing; mammalian cells; site-directed mutagenesis.

MeSH terms

  • Acidaminococcus / genetics
  • Acidaminococcus / metabolism
  • Animals
  • CRISPR-Cas Systems*
  • DNA / metabolism
  • Endonucleases* / metabolism
  • Gene Editing
  • Humans
  • Mammals / metabolism
  • Ruminococcus

Substances

  • DNA
  • Endonucleases

Supplementary concepts

  • Ruminococcus bromii

Grants and funding

R.V.: N.G., A.V., A.K., and M.P. were supported by grants from the Ministry of Science and Higher Education of the Russian Federation allocated to the Kurchatov Center for Genome Research (agreement number 075-15-2019-1659). RS, NN, and IM were supported by the Russian Foundation for Basic Research (19-29-04101). S.T. was supported by the Russian Foundation for Basic Research (20-04-60190). R.V. and P.K. are members of the Moscow University Science and Education School “Molecular Technologies of the Living Systems and Synthetic Biology”. The work was partly done on the equipment purchased in the frame of the Moscow University Program of Development.