Development of an orthogonal CRISPR-Cas transposase system for in situ microbial genome editing

Type I-F CRISPR-associated transposon systems have recently been employed for precise, CRISPR RNA-guided insertion of genetic cargo into a variety of chromosomal targets in diverse bacterial species. Although the characterization of novel type I-F CRISPR-associated transposons often involved multi-plasmid assays, the consolidation of these vectors into a single conjugative DNA-editing all-in-one RNA-guided transposase (DART) plasmid has enabled microbial genome editing in a community setting. We demonstrate here the use of transposon Tn7479, derived from Psychromonas sp. RZ5, as a novel DART system (PsyDART). Importantly, previous characterization of Tn7479 revealed orthogonality to the original DART system, VcDART, with respect to transposon end recognition and mobilization. Before use as a DART system, Tn7479 was reconstituted as a two-plasmid system and tested with a variety of naturally occurring CRISPR repeat variants to determine the optimal CRISPR RNA sequence for RNA-guided transposition. Additionally, truncated versions of the right and left transposon ends were tested to determine minimal transposon end lengths that retained efficient transposition activity. The optimal CRISPR repeats and truncated transposon ends were used to construct a conjugative suicide PsyDART vector, and validation testing was performed in E. coli BL21(DE3). Using an optimized protocol, conjugative delivery by two E. coli strains (WM3036 and MFDpir⁺) resulted in efficient insertion of the associated transposon, encoding an antibiotic resistance gene, at multiple target sites. PsyDART was designed as a broad host range plasmid and future work will be directed towards editing diverse hosts in synthetic soil microbial communities.