Structural basis of RNA-guided DNA integration by type I CRISPR-associated transposases

Area of Science:

• Molecular Biology
• Genetics
• Structural Biology

Background:

• CRISPR-associated transposases (CASTs) integrate DNA site-specifically by combining CRISPR-Cas targeting with transpososome complex assembly.
• Understanding CAST mechanisms is crucial for developing programmable genetic tools, but gaps exist in knowledge of transpososome assembly and activation.
• The Pseudoalteromonas CAST (Pse CAST) system shows high activity in mammalian cells, yet rational engineering is limited by mechanistic uncertainties.

Purpose of the Study:

• To elucidate the stepwise mechanism of RNA-guided DNA integration by the Pse CAST system.
• To visualize functional states of CAST transpososome assembly and activation using structural biology.
• To provide a mechanistic blueprint for engineering improved DNA insertion systems for genome editing.

Main Methods:

• Cryo-electron microscopy (cryo-EM) structural analysis of the Pse CAST system in various functional states.
• DNA transposition assays to validate structural findings and confirm functional mechanisms.
• Detailed structural visualization of target DNA-bound complexes and the complete transpososome holocomplex.

Main Results:

• Cryo-EM structures reveal conformational changes during R-loop formation, target DNA stabilization, and TnsC heptamerization.
• The TnsAB transposase is recruited via interactions with the TnsC C-terminal tail.
• Structures of the Pse CAST holocomplex show TnsC-TnsB and TnsB-target DNA interactions that activate donor DNA integration.

Conclusions:

• Established a unified structural and mechanistic blueprint for RNA-guided DNA integration by CAST systems.
• Identified key interactions and conformational changes governing transpososome assembly and activation.
• Laid the foundation for engineering next-generation DNA insertion systems for advanced genome editing applications.