A thermostable Cas9 with increased lifetime in human plasma
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.
- Authors:
-
- Univ. of California, Berkeley, CA (United States)
- USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
- Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1543743
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Communications
- Additional Journal Information:
- Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2041-1723
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Science & Technology; Other Topics
Citation Formats
Harrington, Lucas B., Paez-Espino, David, Staahl, Brett T., Chen, Janice S., Ma, Enbo, Kyrpides, Nikos C., and Doudna, Jennifer A. A thermostable Cas9 with increased lifetime in human plasma. United States: N. p., 2017.
Web. doi:10.1038/s41467-017-01408-4.
Harrington, Lucas B., Paez-Espino, David, Staahl, Brett T., Chen, Janice S., Ma, Enbo, Kyrpides, Nikos C., & Doudna, Jennifer A. A thermostable Cas9 with increased lifetime in human plasma. United States. https://doi.org/10.1038/s41467-017-01408-4
Harrington, Lucas B., Paez-Espino, David, Staahl, Brett T., Chen, Janice S., Ma, Enbo, Kyrpides, Nikos C., and Doudna, Jennifer A. Fri .
"A thermostable Cas9 with increased lifetime in human plasma". United States. https://doi.org/10.1038/s41467-017-01408-4. https://www.osti.gov/servlets/purl/1543743.
@article{osti_1543743,
title = {A thermostable Cas9 with increased lifetime in human plasma},
author = {Harrington, Lucas B. and Paez-Espino, David and Staahl, Brett T. and Chen, Janice S. and Ma, Enbo and Kyrpides, Nikos C. and Doudna, Jennifer A.},
abstractNote = {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.},
doi = {10.1038/s41467-017-01408-4},
journal = {Nature Communications},
number = 1,
volume = 8,
place = {United States},
year = {2017},
month = {11}
}
Web of Science
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