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Structures of the holo CRISPR RNA-guided transposon integration complex

Author

Listed:
  • Jung-Un Park

    (Cornell University)

  • Amy Wei-Lun Tsai

    (Cornell University)

  • Alexandrea N. Rizo

    (Cornell University)

  • Vinh H. Truong

    (Cornell University)

  • Tristan X. Wellner

    (Cornell University)

  • Richard D. Schargel

    (Cornell University)

  • Elizabeth H. Kellogg

    (Cornell University)

Abstract

CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism1–3. 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 (ShCAST) 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 ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.

Suggested Citation

  • Jung-Un Park & Amy Wei-Lun Tsai & Alexandrea N. Rizo & Vinh H. Truong & Tristan X. Wellner & Richard D. Schargel & Elizabeth H. Kellogg, 2023. "Structures of the holo CRISPR RNA-guided transposon integration complex," Nature, Nature, vol. 613(7945), pages 775-782, January.
  • Handle: RePEc:nat:nature:v:613:y:2023:i:7945:d:10.1038_s41586-022-05573-5
    DOI: 10.1038/s41586-022-05573-5
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    Cited by:

    1. Yunha Hwang & Andre L. Cornman & Elizabeth H. Kellogg & Sergey Ovchinnikov & Peter R. Girguis, 2024. "Genomic language model predicts protein co-regulation and function," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    2. Mercedes Spínola-Amilibia & Lidia Araújo-Bazán & Álvaro Gándara & James M. Berger & Ernesto Arias-Palomo, 2023. "IS21 family transposase cleaved donor complex traps two right-handed superhelical crossings," Nature Communications, Nature, vol. 14(1), pages 1-14, December.

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