30GT

Cryo-EM structure of the PseCascade-TniQ-TnsC complex

Summary for 30GT

• Entry DOI: 10.2210/pdb30gt/pdb
• EMDB information: 57765
• Descriptor: CRISPR RNA, MAGNESIUM ION, TS, ... (10 entities in total)
• Functional Keywords: transposase, dna-binding, rna-binding, crispr-cas, crispr-associated transposon, dna binding protein
• Biological source: Pseudoalteromonas sp. S983; More
• Total number of polymer chains: 20
• Total formula weight: 822533.65

Authors

Finocchio, G.,Oberli, S.,Schmitz, M.,Jinek, M. (deposition date: 2026-04-24, release date: 2026-06-17)

Primary citation

Finocchio, G.,Oberli, S.,Lampe, G.,Schmitz, M.,Sternberg, S.H.,Jinek, M.
Structural basis of RNA-guided DNA integration by type I CRISPR-associated transposases.
Biorxiv, 2026

PubMed Abstract: 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.

PubMed: 42239233
DOI: 10.64898/2026.05.18.725949

Experimental method

ELECTRON MICROSCOPY (3.3 Å)