Abstract
Genome engineering tools in bacteria are limited by their targeting abilities, cargo size capacities, and integration efficiencies. Programmable Cas-directed transposons have been shown to bypass these constraints; however, genome integrations with these Cas-directed transposons require a cargo plasmid carrying the desired DNA payload flanked by directed repeat transposon arms. This cloning pre-requisite significantly hinders the modularity and streamlining capabilities of Cas-directed transposon systems, diminishing their utility for genome engineering. Here, we present a system that can robustly integrate a linear DNA payload into the genome of E. coli by employing a Type I-F CRISPR-associated transposon from Vibrio cholerae. This system bypasses the traditional limiting factors of Cas-directed transposons by leveraging oligonucleotide design and nested polymerase chain reactions to reconstitute the transposon arms onto a designated cargo. Our findings demonstrate that this programmable linear integration method has high efficiencies in integrating large DNA payloads across multiple genomic loci.
Competing Interest Statement
The authors have declared no competing interest.
