A thermostable Cas9 with increased lifetime in human plasma

Nat Commun. 2017 Nov 10;8(1):1424. doi: 10.1038/s41467-017-01408-4.

Abstract

CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas9 is active at temperatures up to 70 °C, compared to 45 °C for Streptococcus pyogenes Cas9 (SpyCas9), which expands the temperature range for CRISPR-Cas9 applications. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP delivery in vivo and expands the temperature range of CRISPR-Cas9.

Publication types

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

MeSH terms

  • Bacterial Proteins / administration & dosage
  • Bacterial Proteins / blood
  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems
  • Endonucleases / administration & dosage
  • Endonucleases / blood
  • Endonucleases / metabolism*
  • Enzyme Stability
  • Gene Editing
  • Geobacillus stearothermophilus / enzymology*
  • Hot Temperature
  • Humans
  • Models, Molecular
  • Protein Engineering
  • Ribonucleoproteins / administration & dosage

Substances

  • Bacterial Proteins
  • Ribonucleoproteins
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases