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Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2

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  • Xue Huang

    (Hefei National Laboratory for Physical Sciences at the Microscales, University of Science and Technology of China
    National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences)

  • Wei Sun

    (National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences)

  • Zhi Cheng

    (National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Minxuan Chen

    (National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Xueyan Li

    (National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

  • Jiuyu Wang

    (National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences)

  • Gang Sheng

    (Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences)

  • Weimin Gong

    (Hefei National Laboratory for Physical Sciences at the Microscales, University of Science and Technology of China
    School of Life Sciences, University of Science and Technology of China)

  • Yanli Wang

    (National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences
    University of Chinese Academy of Sciences)

Abstract

To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2–crRNA–DNA complexes and a Cas12i2–crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1–8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9–15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.

Suggested Citation

  • Xue Huang & Wei Sun & Zhi Cheng & Minxuan Chen & Xueyan Li & Jiuyu Wang & Gang Sheng & Weimin Gong & Yanli Wang, 2020. "Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2," Nature Communications, Nature, vol. 11(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-19072-6
    DOI: 10.1038/s41467-020-19072-6
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    Cited by:

    1. Colin McGaw & Anthony J. Garrity & Gabrielle Z. Munoz & Jeffrey R. Haswell & Sejuti Sengupta & Elise Keston-Smith & Pratyusha Hunnewell & Alexa Ornstein & Mishti Bose & Quinton Wessells & Noah Jakimo , 2022. "Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing," Nature Communications, Nature, vol. 13(1), pages 1-11, December.

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