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-Structure paper
Title | Structural basis for the assembly of the type V CRISPR-associated transposon complex. |
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Journal, issue, pages | Cell, Vol. 185, Issue 26, Page 4999-5010.e17, Year 2022 |
Publish date | Dec 22, 2022 |
Authors | Michael Schmitz / Irma Querques / Seraina Oberli / Christelle Chanez / Martin Jinek / |
PubMed Abstract | CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the ...CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads. |
External links | Cell / PubMed:36435179 / PubMed Central |
Methods | EM (single particle) |
Resolution | 3.3 - 4.1 Å |
Structure data | EMDB-15974, PDB-8bd4: EMDB-15975, PDB-8bd5: EMDB-15976: Cas12k-sgRNA-dsDNA-TnsC non-productive complex |
Chemicals | ChemComp-ATP: ChemComp-MG: ChemComp-ZN: |
Source |
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Keywords | DNA BINDING PROTEIN / Transposition / TniQ / TnsC / CRISPR-Cas / Tn7-like transposon / RNA BINDING PROTEIN / Cas12k / sgRNA / S15 / Tn7-like transposons |