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Cited by 35CRISPR and Genetic EngineeringRNA regulation and diseaseRNA Interference and Gene Delivery

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

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Deng, Xiangyu, Osikpa, Emmanuel, Yang, Jie, Oladeji, Seye J., Smith, Jamie, Gao, Xue, Gao, Yang·2023·Nature Communications
DOI 10.1038/s41467-023-41501-5PMC PMC10511502
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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.

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  • The highest number of DEGs was from the Cas13bt3-NT sgRNA group compared to Cas13bt3 only or Cas13bt3-VEGFA sgRNA (81 vs 27 and 23).

    “Interestingly, we noted the highest number of differentially expressed genes (DEGs) from the Cas13bt3-NT sgRNA group compared to Cas13bt3 only or Cas13bt3-VEGFA sgRNA (81 vs 27 and 23, respectively; Fig. 1I), a finding that corresponds with recent studies (16, 22).”

    From Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina (2024) · cites this paper as [16]

    The Results section (‘Structural-guided engineering of Cas13bt3’) reports collateral RNA degradation in mammalian cells, which supports a general connection to unintended Cas13bt3 effects. It does not report differentially expressed gene counts or compare a non-targeting sgRNA group with Cas13bt3 alone or a VEGFA-targeting group. The 81, 27, and 23 counts are the citing authors’ finding, not a result established by this source. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Initial in vitro studies show promising efficiency and safety of Cas13bt3.

    “however initial in vitro studies show promising efficiency and safety (13, 16).”

    From Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina (2024) · cites this paper as [16]

    The Results section, “Structural-guided engineering of Cas13bt3” (page unavailable), reports that the Y647A variant retained substantial on-target cleavage while greatly reducing collateral cleavage in biochemical assays; cellular assays also showed robust on-target activity with minimal collateral effects. That supports a promising early efficiency and safety-related finding for the engineered variant, but not for Cas13bt3 generally: the same section reports significant collateral degradation with wild-type Cas13bt3, and it does not establish safety beyond these assays. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

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Cited by

Sorted by citations

Authors
Deng, X., Osikpa, E., Yang, J., Oladeji, S. J., Smith, J., Gao, X., & Gao, Y.
Affiliations
Rice University (US)
Corresponding author
  • Gao, Xue
  • Gao, Yang
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2023-09-20
Volume
14
Issue
1
Pages
5845-5845
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-023-41501-5
PMC
PMC10511502
OpenAlex
W4386887866
Cited by
35
References
3
Citations by year
2026: 12 · 2025: 11 · 2024: 11 · 2023: 1
FWCI
3.12
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Rice Research Repository (Rice University) (submittedVersion, cc-by)
Funders
  • Welch Foundation (C-2033-20200401)
  • Cancer Prevention and Research Institute of Texas (RR190046)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
CRISPR-Cas13, adeno-associated virus delivery, cryo-EM structure, RNA knockdown
MeSH terms
Dependovirus, Endonucleases, RNA, Cryoelectron Microscopy, CRISPR-Cas Systems

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