A Thermostable Cas9 with Increased Lifetime in Human Plasma
Open accessPublished version of a preprint
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.
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GeoCas9 showed editing efficiency comparable to SpCas9 when used for disruption of GFP function in GFP-expressing HEK293T cells.
“In GFP-expressing HEK293T cells, GeoCas9 showed editing efficiency comparable to SpCas9 when used for the disruption of GFP function [97].”
From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [97]
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References 57 · Cited by 205
Research works printed but not linked: 1
- [3]Chylinski K Makarova KS Charpentier E Koonin EV Classification and evolution of type II CRISPR-Cas systems Nucleic Acids Res. 2014 42 6091 6105 10.1093/nar/gku241 24728998 PMC4041416 · DOI · Paper page
- Authors
- Harrington, L. B., Paez-Espino, D., Staahl, B. T., Chen, J. S., Ma, E., Kyrpides, N. C., & Doudna, J. A.
- Affiliations
- University of California, Berkeley (US)
- Joint Genome Institute (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Doudna, Jennifer A.
- Venue
- Nature Communications
- Published
- 2017-11-10
- Volume
- 8
- Issue
- 1
- Pages
- 1424-1424
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC5681539
- OpenAlex
- W2949986034
- Cited by
- 205
- References
- 57
- Citations by year
- 2026: 10 · 2025: 30 · 2024: 21 · 2023: 23 · 2022: 29 · 2021: 22 · 2020: 26 · 2019: 16 · 2018: 25 · 2017: 3
- FWCI
- 7.82
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Science Foundation (1244557)
- Howard Hughes Medical Institute
- Paul G. Allen Frontiers Group
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- Bacillus stearothermophilus, CRISPR/Cas9, RNA-guided DNA cleavage, thermophilic bacteria, ribonucleoprotein complexes, in vivo gene editing, SpCas9, genome editing
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, Geobacillus stearothermophilus, Bacterial Proteins, Endonucleases, Enzyme Stability, Hot Temperature, Humans, Models, Molecular, Ribonucleoproteins, Protein Engineering, CRISPR-Cas Systems
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