30GA

Cryo-EM structure of the PseCascade-TniQ-TnsC-TnsAB holocomplex

Summary for 30GA

• Entry DOI: 10.2210/pdb30ga/pdb
• EMDB information: 57736
• Descriptor: CRISPR RNA, Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase, MAGNESIUM ION, ... (12 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: 35
• Total formula weight: 2492176.44

Authors

Finocchio, G.,Oberli, S.,Schmitz, M.,Jinek, M. (deposition date: 2026-04-23, 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 (3.2 Å)