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TitleStructural basis of RNA-guided DNA integration by type I CRISPR-associated transposases.
Journal, issue, pagesbioRxiv, Year 2026
Publish dateMay 18, 2026
AuthorsGiada Finocchio / Seraina Oberli / George Lampe / Michael Schmitz / Samuel H Sternberg / Martin Jinek
PubMed AbstractCRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like ...CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex . Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools . The type I-F CAST ( CAST) displays the highest activity in mammalian cells to date and has been the subject of extensive directed evolution , but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation . Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the CAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa CAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.
External linksbioRxiv / PubMed:42239233 / PubMed Central
MethodsEM (single particle)
Resolution2.8 - 3.4 Å
Structure data

EMDB-57728: TnsAB-focused cryo-EM volume of the PseCascade-TniQ-TnsC-TnsAB holocomplex
Method: EM (single particle) / Resolution: 3.4 Å

EMDB-57729: TnsC-focused cryo-EM volume of the PseCascade-TniQ-TnsC-TnsAB holocomplex
Method: EM (single particle) / Resolution: 3.3 Å

EMDB-57730: Cascade-TniQ-TnsC-focused cryo-EM volume of the PseCascade-TniQ-TnsC-TnsAB holocomplex
Method: EM (single particle) / Resolution: 3.1 Å

EMDB-57731: Consensus cryo-EM volume of the PseCascade-TniQ-TnsC-TnsAB holocomplex
Method: EM (single particle) / Resolution: 3.1 Å

EMDB-57736, PDB-30ga:
Cryo-EM structure of the PseCascade-TniQ-TnsC-TnsAB holocomplex
Method: EM (single particle) / Resolution: 3.2 Å

EMDB-57737: TnsC-focused cryo-EM volume of the PseCascade-TniQ-TnsC complex bound to TnsB-hook motifs
Method: EM (single particle) / Resolution: 3.0 Å

EMDB-57738: Consensus cryo-EM volume of the PseCascade-TniQ-TnsC complex bound to TnsB-hook motifs
Method: EM (single particle) / Resolution: 2.8 Å

EMDB-57739, PDB-30gb:
Cryo-EM structure of the PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs
Method: EM (single particle) / Resolution: 2.9 Å

EMDB-57750: TnsC-focused cryo-EM volume of the PseCascade-TniQ-TnsC complex
Method: EM (single particle) / Resolution: 3.4 Å

EMDB-57751: Consensus cryo-EM volume of the PseCascade-TniQ-TnsC complex
Method: EM (single particle) / Resolution: 3.1 Å

EMDB-57757: Cryo-EM volume of the PseCascade-TniQ complex
Method: EM (single particle) / Resolution: 3.2 Å

EMDB-57758: Cryo-EM volume of the PseCascade complex
Method: EM (single particle) / Resolution: 2.9 Å

EMDB-57765, PDB-30gt:
Cryo-EM structure of the PseCascade-TniQ-TnsC complex
Method: EM (single particle) / Resolution: 3.3 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

Source
  • pseudoalteromonas sp. s983 (bacteria)
  • escherichia coli k-12 (bacteria)
KeywordsDNA BINDING PROTEIN / Transposase / DNA-binding / RNA-binding / CRISPR-Cas / CRISPR-associated transposon

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