Structures of the holo CRISPR RNA-guided transposon integration complex
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Abstract
CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism 1–3 . CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA–TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii ( Sh CAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC–TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein–DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This Sh CAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
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- Authors
- Park, J.-U., Tsai, A. W.-L., Rizo, A. N., Truong, V. H., Wellner, T. X., Schargel, R. D., & Kellogg, E. H.
- Affiliations
- Cornell University (US)
- Corresponding author
- Kellogg, Elizabeth H.
- ORCID
- Venue
- Nature
- Published
- 2022-11-28
- Volume
- 613
- Issue
- 7945
- Pages
- 775-782
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC9876797
- OpenAlex
- W4310155672
- Cited by
- 80
- References
- 59
- Citations by year
- 2026: 22 · 2025: 21 · 2024: 21 · 2023: 15 · 2022: 1
- FWCI
- 4.74
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Nature (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Funders
- Simons Foundation (SF 349247)
- National Institutes of Health (1s10od030470-01, MCB200090)
- National Institute of General Medical Sciences (U24GM129539, R01GM144566)
- Common Fund (U24 GM129539)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Keywords
- transpososome, cascades, ribosomal protein S15, R-loops, protein-DNA interactions, transposition mechanism, genome editing
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Cyanobacteria, Bacterial Proteins, DNA Transposable Elements, DNA-Binding Proteins, Endodeoxyribonucleases, Transposases, Holoenzymes, Cryoelectron Microscopy, Multiprotein Complexes, Ribosome Subunits, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, CRISPR-Associated Proteins
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