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A thermostable Cas9 with increased lifetime in human plasma

Author

Listed:
  • Lucas B. Harrington

    (University of California)

  • David Paez-Espino

    (Joint Genome Institute)

  • Brett T. Staahl

    (University of California)

  • Janice S. Chen

    (University of California)

  • Enbo Ma

    (University of California)

  • Nikos C. Kyrpides

    (Joint Genome Institute)

  • Jennifer A. Doudna

    (University of California
    University of California
    University of California
    University of California)

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.

Suggested Citation

  • Lucas B. Harrington & David Paez-Espino & Brett T. Staahl & Janice S. Chen & Enbo Ma & Nikos C. Kyrpides & Jennifer A. Doudna, 2017. "A thermostable Cas9 with increased lifetime in human plasma," Nature Communications, Nature, vol. 8(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_s41467-017-01408-4
    DOI: 10.1038/s41467-017-01408-4
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    Cited by:

    1. Zhiheng Yang & Zilong Li & Bixiao Li & Ruihong Bu & Gao-Yi Tan & Zhengduo Wang & Hao Yan & Zhenguo Xin & Guojian Zhang & Ming Li & Hua Xiang & Lixin Zhang & Weishan Wang, 2023. "A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    2. Marcus A. Toral & Carsten T. Charlesworth & Benjamin Ng & Teja Chemudupati & Shota Homma & Hiromitsu Nakauchi & Alexander G. Bassuk & Matthew H. Porteus & Vinit B. Mahajan, 2022. "Investigation of Cas9 antibodies in the human eye," Nature Communications, Nature, vol. 13(1), pages 1-9, December.

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