Target site selection and remodelling by type V CRISPR-transposon systems

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• CRISPR-Cas systems offer adaptive immunity against mobile genetic elements.
• Tn7-like transposons utilize CRISPR systems (types I-F, I-B, V-K) for RNA-guided DNA insertion.
• The molecular mechanism of RNA-directed DNA transposition by type V-K CRISPR-associated transposons remains unclear.

Purpose of the Study:

• To elucidate the molecular mechanism of RNA-directed DNA transposition by type V-K CRISPR-associated transposons.
• To determine the structural basis of target DNA recognition by the Cas12k-guide RNA complex.
• To understand the role of TnsC, TnsB, and TniQ in the transposition process.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structures of Cas12k-guide RNA-target DNA complex and DNA-bound TnsC filament.
• In vivo transposition assays to validate structural findings.
• Biochemical experiments to investigate protein interactions and enzymatic activities.

Main Results:

• Cryo-EM structures revealed intricate guide RNA architecture and critical interactions for RNA-guided DNA recognition by Cas12k.
• TnsC filament assembly is ATP-dependent and induces DNA duplex remodelling.
• TniQ restricts TnsC polymerization, while TnsB triggers TnsC filament disassembly upon ATP hydrolysis, facilitating transposon insertion.

Conclusions:

• RNA-directed target selection by Cas12k initiates TnsC polymerization and DNA remodelling.
• This process creates a platform for TnsB to catalyze site-specific transposon insertion.
• Findings provide insights for developing CRISPR-associated transposons as programmable gene insertion tools.