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Structural basis for the activation of a compact CRISPR-Cas13 nuclease

Author

Listed:
  • Xiangyu Deng

    (Rice University)

  • Emmanuel Osikpa

    (Rice University)

  • Jie Yang

    (Rice University)

  • Seye J. Oladeji

    (Rice University)

  • Jamie Smith

    (Rice University)

  • Xue Gao

    (Rice University
    Rice University
    Rice University)

  • Yang Gao

    (Rice University)

Abstract

The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal “UC” sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications.

Suggested Citation

  • Xiangyu Deng & Emmanuel Osikpa & Jie Yang & Seye J. Oladeji & Jamie Smith & Xue Gao & Yang Gao, 2023. "Structural basis for the activation of a compact CRISPR-Cas13 nuclease," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-41501-5
    DOI: 10.1038/s41467-023-41501-5
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    References listed on IDEAS

    as
    1. Alexandra East-Seletsky & Mitchell R. O’Connell & Spencer C. Knight & David Burstein & Jamie H. D. Cate & Robert Tjian & Jennifer A. Doudna, 2016. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection," Nature, Nature, vol. 538(7624), pages 270-273, October.
    2. Omar O. Abudayyeh & Jonathan S. Gootenberg & Patrick Essletzbichler & Shuo Han & Julia Joung & Joseph J. Belanto & Vanessa Verdine & David B. T. Cox & Max J. Kellner & Aviv Regev & Eric S. Lander & Da, 2017. "RNA targeting with CRISPR–Cas13," Nature, Nature, vol. 550(7675), pages 280-284, October.
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