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Structure paper

TitleStructural basis for the assembly of the type V CRISPR-associated transposon complex.
Journal, issue, pagesCell, Vol. 185, Issue 26, Page 4999-5010.e17, Year 2022
Publish dateDec 22, 2022
AuthorsMichael Schmitz / Irma Querques / Seraina Oberli / Christelle Chanez / Martin Jinek /
PubMed AbstractCRISPR-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 linksCell / PubMed:36435179 / PubMed Central
MethodsEM (single particle)
Resolution3.3 - 4.1 Å
Structure data

15974

8bd4

EMDB-15974, PDB-8bd4:
TniQ-capped Tns-ATP-dsDNA complex
Method: EM (single particle) / Resolution: 3.44 Å

15975

8bd5

EMDB-15975, PDB-8bd5:
Cas12k-sgRNA-dsDNA-S15-TniQ-TnsC transposon recruitment complex
Method: EM (single particle) / Resolution: 3.3 Å

15976

8bd6

EMDB-15976: Cas12k-sgRNA-dsDNA-TnsC non-productive complex
PDB-8bd6: Cas12k-sgRNA-dsDNA-TnsC non-productive complex.
Method: EM (single particle) / Resolution: 4.1 Å

Chemicals

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

ChemComp-MG:
Unknown entry

ChemComp-ZN:
Unknown entry

Source
  • scytonema hofmannii (bacteria)
  • synthetic construct (others)
  • escherichia coli k-12 (bacteria)
KeywordsDNA BINDING PROTEIN / Transposition / TniQ / TnsC / CRISPR-Cas / Tn7-like transposon / RNA BINDING PROTEIN / Cas12k / sgRNA / S15 / Tn7-like transposons

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