RT Journal Article
SR Electronic
T1 A thermostable Cas9 with increased lifetime in human plasma
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 138867
DO 10.1101/138867
A1 Lucas B. Harrington
A1 David Paez-Espino
A1 Janice S. Chen
A1 Enbo Ma
A1 Brett T. Staahl
A1 Nikos C. Kyrpides
A1 Jennifer Doudna
YR 2017
UL http://biorxiv.org/content/early/2017/05/16/138867.abstract
AB 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 proteolytic degradation and unsuitable for applications requiring function at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage over a wide temperature range and has an enhanced protein lifetime in human plasma. GeoCas9 is active at temperatures up to 70°C, compared to 45°C for Streptococcus pyogenes Cas9 (SpyCas9), which greatly expands the temperature range for CRISPR-Cas9 applications. By comparing features of two closely related Geobacillus homologs, we created a variant of GeoCas9 that doubles the DNA target sequences that can be recognized by this system. 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.