A thermostable Cas9 with increased lifetime in human plasma

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....

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Bibliographic Details
Published in:	Nature communications 2017-11, Vol.8 (1), p.1424-8, Article 1424
Main Authors:	Harrington, Lucas B, Paez-Espino, David, Staahl, Brett T, Chen, Janice S, Ma, Enbo, Kyrpides, Nikos C, Doudna, Jennifer A
Format:	Article
Language:	eng
Subjects:	Amino acids Bacteria Bacterial Proteins - administration & dosage Bacterial Proteins - blood Bacterial Proteins - metabolism BASIC BIOLOGICAL SCIENCES Biodegradation Blood plasma Cleavage CRISPR CRISPR-Associated Protein 9 CRISPR-Cas Systems Deoxyribonucleic acid DNA Editing Endonucleases - administration & dosage Endonucleases - blood Endonucleases - metabolism Enzyme Stability Enzymes Gene Editing Genomes Geobacillus stearothermophilus - enzymology Homology Hot Temperature Humans Mesophilic bacteria Metabolism Models, Molecular Other Topics Protein Engineering Proteins Ribonucleic acid Ribonucleoproteins - administration & dosage RNA Science & Technology Temperature Thermophilic bacteria
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Summary:	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.
ISSN:	2041-1723 2041-1723