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A thermostable Cas9 with increased lifetime in human plasma

Lucas B. Harrington, David Paez-Espino, Janice S. Chen, Enbo Ma, Brett T. Staahl, Nikos C. Kyrpides, Jennifer Doudna
doi: https://doi.org/10.1101/138867
Lucas B. Harrington
1Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA.
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David Paez-Espino
2Department of Energy, Joint Genome Institute, Walnut Creek, California 94598, USA.
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Janice S. Chen
1Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA.
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Enbo Ma
1Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA.
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Brett T. Staahl
1Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA.
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Nikos C. Kyrpides
2Department of Energy, Joint Genome Institute, Walnut Creek, California 94598, USA.
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Jennifer Doudna
1Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA.
3Department of Chemistry, University of California, Berkeley, California, 94720, USA.
4Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
5Innovative Genomics Institute, University of California, Berkeley, California 94720, USA.
6MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
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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 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.

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Posted May 16, 2017.
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A thermostable Cas9 with increased lifetime in human plasma
Lucas B. Harrington, David Paez-Espino, Janice S. Chen, Enbo Ma, Brett T. Staahl, Nikos C. Kyrpides, Jennifer Doudna
bioRxiv 138867; doi: https://doi.org/10.1101/138867
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A thermostable Cas9 with increased lifetime in human plasma
Lucas B. Harrington, David Paez-Espino, Janice S. Chen, Enbo Ma, Brett T. Staahl, Nikos C. Kyrpides, Jennifer Doudna
bioRxiv 138867; doi: https://doi.org/10.1101/138867

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