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CasX enzymes comprise a distinct family of RNA-guided genome editors

Author

Listed:
  • Jun-Jie Liu

    (University of California
    University of California
    Lawrence Berkeley National Laboratory)

  • Natalia Orlova

    (University of California)

  • Benjamin L. Oakes

    (University of California)

  • Enbo Ma

    (University of California)

  • Hannah B. Spinner

    (University of California)

  • Katherine L. M. Baney

    (University of California)

  • Jonathan Chuck

    (University of California)

  • Dan Tan

    (Salk Institute for Biological Studies)

  • Gavin J. Knott

    (University of California)

  • Lucas B. Harrington

    (University of California)

  • Basem Al-Shayeb

    (University of California)

  • Alexander Wagner

    (Max-Planck-Institute for Biochemistry)

  • Julian Brötzmann

    (Ludwig-Maximilians-University)

  • Brett T. Staahl

    (University of California
    University of California)

  • Kian L. Taylor

    (University of California)

  • John Desmarais

    (University of California)

  • Eva Nogales

    (University of California
    University of California
    Lawrence Berkeley National Laboratory
    University of California)

  • Jennifer A. Doudna

    (University of California
    Lawrence Berkeley National Laboratory
    University of California
    University of California)

Abstract

The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR–CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.

Suggested Citation

  • Jun-Jie Liu & Natalia Orlova & Benjamin L. Oakes & Enbo Ma & Hannah B. Spinner & Katherine L. M. Baney & Jonathan Chuck & Dan Tan & Gavin J. Knott & Lucas B. Harrington & Basem Al-Shayeb & Alexander W, 2019. "CasX enzymes comprise a distinct family of RNA-guided genome editors," Nature, Nature, vol. 566(7743), pages 218-223, February.
    • Handle: RePEc:nat:nature:v:566:y:2019:i:7743:d:10.1038_s41586-019-0908-x
    DOI: 10.1038/s41586-019-0908-x
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    Cited by:

    1. Changchang Xin & Jianhang Yin & Shaopeng Yuan & Liqiong Ou & Mengzhu Liu & Weiwei Zhang & Jiazhi Hu, 2022. "Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    2. Greta Bigelyte & Joshua K. Young & Tautvydas Karvelis & Karolina Budre & Rimante Zedaveinyte & Vesna Djukanovic & Elizabeth Ginkel & Sushmitha Paulraj & Stephen Gasior & Spencer Jones & Lanie Feigenbu, 2021. "Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells," Nature Communications, Nature, vol. 12(1), pages 1-8, December.
    3. Raed Ibraheim & Phillip W. L. Tai & Aamir Mir & Nida Javeed & Jiaming Wang & Tomás C. Rodríguez & Suk Namkung & Samantha Nelson & Eraj Shafiq Khokhar & Esther Mintzer & Stacy Maitland & Zexiang Chen &, 2021. "Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo," Nature Communications, Nature, vol. 12(1), pages 1-17, December.

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