Structures of the holo CRISPR RNA-guided transposon integration complex

Area of Science:

• Molecular Biology
• Genetics
• Structural Biology

Background:

• CRISPR-associated transposons (CAST) are mobile genetic elements that use RNA-guided mechanisms to insert large DNA payloads.
• CAST systems comprise multiple conserved proteins, including a CRISPR effector, a AAA+ regulator, a transposase, and a target-site-associated factor, forming a transposition integration complex (transpososome).

Purpose of the Study:

• To reconstitute and determine the high-resolution structure of the type V-K CAST transpososome from Scytonema hofmannii (ShCAST).
• To elucidate the molecular architecture and protein-DNA interactions within the ShCAST transpososome, providing mechanistic insights into CAST transposition.

Main Methods:

• Single-particle cryo-electron microscopy (cryo-EM) was employed to determine the structure of the approximately 1 MDa ShCAST transpososome.
• Biochemical assays were used to investigate protein-protein and protein-DNA interactions and their impact on transposition activity.

Main Results:

• The cryo-EM structure revealed a modular organization of the ShCAST transpososome, detailing interactions between Cas12k, TniQ, TnsC, and TnsB.
• Cas12k stabilizes an R-loop structure, while TnsC interacts with TniQ and TnsB, with TnsC-TnsB interactions stimulating ATPase activity.
• The structure shows altered TnsC-bound DNA conformation and new protein-DNA interactions crucial for transposition, along with evidence of flexible CRISPR effector association.

Conclusions:

• The determined ShCAST transpososome structure provides unprecedented detail on the assembly and function of type V-K CAST systems.
• Understanding these molecular mechanisms opens avenues for engineering CAST systems for enhanced precision in genome editing applications.