Mechanism of target site selection by type V-K CRISPR-associated transposases

Science. 2023 Nov 17;382(6672):eadj8543. doi: 10.1126/science.adj8543. Epub 2023 Nov 17.

Abstract

CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo-electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.

MeSH terms

  • CRISPR-Associated Proteins* / genetics
  • CRISPR-Cas Systems*
  • Cryoelectron Microscopy
  • Cyanobacteria / enzymology
  • DNA Transposable Elements*
  • Gene Editing* / methods
  • Single Molecule Imaging
  • Transposases* / genetics
  • Transposases* / metabolism

Substances

  • CRISPR-Associated Proteins
  • DNA Transposable Elements
  • Transposases