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

A thermostable Cas9 with increased lifetime in human plasma.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA.
2
Department of Energy, Joint Genome Institute, Walnut Creek, CA, 94598, USA.
3
Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA. doudna@berkeley.edu.
4
Department of Chemistry, University of California, Berkeley, CA, 94720, USA. doudna@berkeley.edu.
5
Howard Hughes Medical Institute, University of California, Berkeley, CA, 94720, USA. doudna@berkeley.edu.
6
Innovative Genomics Institute, University of California, Berkeley, CA, 94720, USA. doudna@berkeley.edu.
7
MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA. doudna@berkeley.edu.

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.

PMID:
29127284
PMCID:
PMC5681539
DOI:
10.1038/s41467-017-01408-4
[Indexed for MEDLINE]
Free PMC Article

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