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.
Highlights
CRISPR-Cas[9] is a powerful technology that has enabled genome editing in a wide range of species
Recent efforts using SpyCas[9] to edit a facultative thermophile have been possible by reducing the temperature within the organism[12]. This approach is not feasible for obligate thermophiles, and requires additional steps for moderate thermophiles. This is especially important for metabolic engineering for which thermophilic bacteria present enticing hosts for chemical synthesis due to decreased risk of contamination, continuous recovery of volatile products, and the ability to conduct reactions that are thermodynamically unfavorable in mesophilic hosts[13]
The Cas[9] from G. stearothermophilus (G. st.; formerly Bacillus stearothermophilus)[18] stood out because it was full-length and its sequence is shorter than the average Cas[9]
Summary
CRISPR-Cas[9] is a powerful technology that has enabled genome editing in a wide range of species. The use of CRISPR-Cas[9] has rapidly transformed the ability to edit and modulate the genomes of a wide range of organisms[1] This technology, derived from adaptive immune systems found in thousands of bacterial species, relies on RNA-guided recognition and cleavage of invasive viral and plasmid DNA2. In addition to SpyCas[9], several other Cas[9] proteins have been shown to edit mammalian genomes with varying efficiencies[5, 7,8,9,10] While these proteins together provide a robust set of tools, they all originate from mesophilic hosts, making them unsuitable for applications requiring cleavage at higher temperatures or extended protein stability.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
