Structural basis for the assembly of the type V CRISPR-associated transposon complex

Area of Science:

• Molecular Biology
• Genetics
• Structural Biology

Background:

• CRISPR-Cas systems are adapted by Tn7-like elements for RNA-guided transposition.
• Type V-K CRISPR-associated transposons utilize Cas12k, TnsC, TniQ, and TnsB for transposition.

Purpose of the Study:

• To elucidate the structural and mechanistic basis of Type V-K CRISPR-associated transposon assembly.
• To identify the components and interactions involved in RNA-guided transposition.

Main Methods:

• Cryo-electron microscopy to determine the structure of the Cas12k-transposon recruitment complex.
• Transposition activity assays to validate structural findings and functional roles.

Main Results:

• A cryo-electron microscopic structure of a target DNA-bound complex revealed the unexpected presence of ribosomal protein S15.
• Complex assembly involves interactions between guide RNA, TniQ, S15, Cas12k, and TnsC, leading to R-loop completion.
• TniQ nucleates TnsC polymerization at the Cas12k-proximal filament end.
• Ribosomal protein S15 is identified as a functional component of the Type V CRISPR-associated transposon machinery.

Conclusions:

• The study uncovers key mechanistic aspects of RNA-mediated assembly in CRISPR-associated transposons.
• The findings highlight the role of S15 in guiding transposition and suggest its potential for developing programmable DNA insertion tools.