Transposon end recognition and excision mechanisms of type I-F CRISPR-associated transposases
Abstract CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for programmable, site-specific integration. Among them, the type I-F system from Ps e udoalteromonas ( Pse CAST) shows exceptionally robust activity in human cells, yet its mechanistic basis remains poorly understood. Here, we present structural and biochemical analysis of the Pse CAST transposase TnsAB. Biochemical reconstitution of transposon DNA excision defines key characteristics of the transposition mechanism. Cryogenic electron microscopy (cryo-EM) structures of Pse TnsAB paired-end complexes reveal molecular determinants of transpososome assembly, transposon end recognition and cleavage. We validate these findings using biochemical and in vivo assays of structure-based transposase mutants, and provide mechanistic insights into the enhanced activity of a laboratory-evolved TnsAB variant. Together, our studies highlight molecular features underlying the efficiency of natural and engineered type I-F transposases and establish a mechanistic framework for their continued rational optimization.
Authors
- Martin Jínek (ORCID: https://orcid.org/0000-0002-7601-210X)
- Seraina Oberli (ORCID: https://orcid.org/0000-0002-3568-7637)
- Giada Finocchio (ORCID: https://orcid.org/0000-0001-6905-9308)
- Irma Querques (ORCID: https://orcid.org/0000-0003-1113-2773)
- George D. Lampe (ORCID: https://orcid.org/0000-0002-2873-2372)
- Thomas Swartjes (ORCID: https://orcid.org/0000-0001-7528-5483)
- Samuel Henry Sternberg (ORCID: https://orcid.org/0000-0001-8240-9114)
- Julia Karan (ORCID: https://orcid.org/0009-0005-8284-7225)
- Mateusz Walter (ORCID: https://orcid.org/0009-0001-7023-4545)
- Iana Camilla Hammerschmid (ORCID: https://orcid.org/0009-0003-6670-6196)
Institutions
- University of Vienna (AT)
- Howard Hughes Medical Institute (US)
- University of Zurich (CH)
- Max Perutz Labs (AT)
- Vienna Biocenter (AT)
- Medical University of Vienna (AT)
- Columbia University (US)
Publication Details
- Journal
- Nature Communications
- Published
- 2026-08-24
- DOI
- https://doi.org/10.1038/s41467-026-76893-7
- Citations
- 1
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 3.12
Funders
- National Science Foundation
- Howard Hughes Medical Institute
- Vallee Foundation
- European Commission
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
- Österreichischen Akademie der Wissenschaften
- Eidgenössische Technische Hochschule Zürich
- Universität Wien
- Medizinische Universität Wien
- National Institutes of Health
- Horizon 2020 Framework Programme
- Irving Medical Center, Columbia University
- HORIZON EUROPE Framework Programme