RT Journal Article
SR Electronic
T1 A universal system for streamlined genome integrations with CRISPR-associated transposases
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.05.30.494051
DO 10.1101/2022.05.30.494051
A1 Wang, Megan
A1 Sanfiorenzo, Charles
A1 Zhang, Raymond J.
A1 Wang, Kaihang
YR 2022
UL http://biorxiv.org/content/early/2022/05/31/2022.05.30.494051.abstract
AB 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 StatementThe authors have declared no competing interest.