Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d
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Abstract
CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
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crRNAs are typically protected from degradation while complexed with Cas13, but the ends of longer bcgRNA molecules may still be accessible and susceptible to degradation.
“While we expect that crRNA are typically protected from degradation while complexed with Cas13 (refs. 31,32), the ends of longer bcgRNA molecules may still be accessible and susceptible to degradation.”
From Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq (2023) · cites this paper as [32]
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Self-citation
- Authors
- Zhang, C., Konermann, S., Brideau, N. J., Lotfy, P., Wu, X., Novick, S. J., Strutzenberg, T., Griffin, P. R., Hsu, P. D., & Lyumkis, D.
- Affiliations
- Salk Institute for Biological Studies (US)
- Whitehead Institute for Biomedical Research (US)
- Scripps Research Institute (US)
- Corresponding author
- Hsu, Patrick D.
- Lyumkis, Dmitry
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2018-09
- Volume
- 175
- Issue
- 1
- Pages
- 212-223.e17
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC6179368
- OpenAlex
- W2798809372
- Cited by
- 276
- References
- 60
- Citations by year
- 2026: 27 · 2025: 28 · 2024: 51 · 2023: 45 · 2022: 41 · 2021: 39 · 2020: 26 · 2019: 17 · 2018: 2
- FWCI
- 7.47
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- NIH-NCI (P30 014195)
- Howard Hughes Medical Institute
- Helen Hay Whitney Foundation
- NIH (DP5 OD021369, DP5 OD021396, U54GM103368)
- Helmsley Charitable Trust
- 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 Research and Splicing
- Keywords
- CRISPR-Cas13d, guide RNA, ternary complex structure, hydrogen-deuterium exchange, conformational dynamics, RNA cleavage mechanism, prokaryotic adaptive immunity
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Endonucleases, Humans, Molecular Conformation, Ribonucleases, RNA, Cryoelectron Microscopy, HEK293 Cells, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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