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Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d

Author

Listed:
  • Bo Zhang

    (Fujian Normal University
    Fujian Normal University
    Pilot National Laboratory for Marine Science and Technology (Qingdao))

  • Yangmiao Ye

    (Fujian Normal University)

  • Weiwei Ye

    (Fujian Normal University)

  • Vanja Perčulija

    (Fujian Normal University)

  • Han Jiang

    (Fujian Normal University)

  • Yiyang Chen

    (Fujian Normal University)

  • Yu Li

    (Fujian Normal University)

  • Jing Chen

    (Fujian Normal University)

  • Jinying Lin

    (Fujian Normal University)

  • Siqi Wang

    (Fujian Normal University)

  • Qi Chen

    (Fujian Normal University)

  • Yu-San Han

    (National Taiwan University)

  • Songying Ouyang

    (Fujian Normal University
    Fujian Normal University
    Pilot National Laboratory for Marine Science and Technology (Qingdao)
    Chinese Academy of Sciences)

Abstract

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3′-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.

Suggested Citation

  • Bo Zhang & Yangmiao Ye & Weiwei Ye & Vanja Perčulija & Han Jiang & Yiyang Chen & Yu Li & Jing Chen & Jinying Lin & Siqi Wang & Qi Chen & Yu-San Han & Songying Ouyang, 2019. "Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-10507-3
    DOI: 10.1038/s41467-019-10507-3
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    Cited by:

    1. Hung-Che Kuo & Joshua Prupes & Chia-Wei Chou & Ilya J. Finkelstein, 2024. "Massively parallel profiling of RNA-targeting CRISPR-Cas13d," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    2. Feiyu Zhao & Tao Zhang & Xiaodi Sun & Xiyun Zhang & Letong Chen & Hejun Wang & Jinze Li & Peng Fan & Liangxue Lai & Tingting Sui & Zhanjun Li, 2023. "A strategy for Cas13 miniaturization based on the structure and AlphaFold," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    3. David N. Fiflis & Nicolas A. Rey & Harshitha Venugopal-Lavanya & Beatrice Sewell & Aaron Mitchell-Dick & Katie N. Clements & Sydney Milo & Abigail R. Benkert & Alan Rosales & Sophia Fergione & Aravind, 2024. "Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    4. Antonios Apostolopoulos & Naohiro Kawamoto & Siu Yu A. Chow & Hitomi Tsuiji & Yoshiho Ikeuchi & Yuichi Shichino & Shintaro Iwasaki, 2024. "dCas13-mediated translational repression for accurate gene silencing in mammalian cells," Nature Communications, Nature, vol. 15(1), pages 1-18, December.

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