Genome editing tools,
through the disruption of an organism’s
native genetic material or the introduction of non-native DNA, facilitate
functional investigations to link genotypes to phenotypes. Transposons
have been instrumental genetic tools in microbiology, enabling genome-wide,
randomized disruption of genes and insertions of new genetic elements.
Due to this randomness, identifying and isolating particular transposon
mutants (i.e., those with modifications at a genetic locus of interest)
can be laborious, often requiring one to sift through hundreds or
thousands of mutants. Programmable, site-specific targeting of transposons
became
possible with recently described CRISPR-associated transposase (CASTs)
systems, allowing the streamlined recovery of desired mutants in a
single step. Like other CRISPR-derived systems, CASTs can be programmed
by guide-RNA that is transcribed from short DNA sequence(s). Here,
we describe a CAST system and demonstrate its function in bacteria
from three classes of Proteobacteria. A dual plasmid strategy is demonstrated:
(i) CAST genes are expressed from a broad-host-range replicative plasmid
and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal
pUC plasmid. Using our CAST system, single-gene disruptions were performed
with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria
(Burkholderia thailandensis and Pseudomonas putida, respectively). We also report
a peak efficiency of 45% in the Alphaproteobacterium Agrobacterium fabrum. In B. thailandensis, we performed simultaneous co-integration of transposons at two
different target sites, demonstrating CAST’s utility in multilocus
strategies. The CAST system is also capable of high-efficiency large
transposon insertion totaling over 11 kbp in all three bacteria tested.
Lastly, the dual plasmid system allowed for iterative transposon mutagenesis
in all three bacteria without loss of efficiency. Given these iterative
capabilities and large payload capacity, this system will be helpful
for genome engineering experiments across several fields of research.