Investigating and Leveraging Type I-F CRISPR-Associated Transposon Regulators to Improve Editing Efficiency and Target Range

Type I-F CRISPR-associated transposons (CASTs) are a newly-discovered tool with unique potential in applying targeted genetic edits to complex microbial communities. CASTs are broadly dispersed across bacteria and capable of integrating large genomic payloads. However, little is known about the host molecular factors which regulate CAST integration and their widespread utilization is limited by low efficiency across diverse, non-model bacteria. To expand the range and applicability of Type I-F CASTs as editing tools, we employed a whole genome mutant screening approach to identify putative regulators of CAST transposition in established model systems in which CASTs are known to integrate. Candidate regulator hits were individually validated, with a particular focus on well-characterized genes involved in known mechanisms. Next, we conducted a bioinformatic survey for the conservation of our candidate regulator hits across broad bacterial phyla. Finally, we leveraged our findings by constructing vectors that incorporate these key regulators to increase host range and editing efficiency. These results will shed light on the molecular mechanisms underlying CAST integration and enable more efficient editing in diverse non-model microorganisms.