UltraCAST: A Flexible All-In-One Suicide Vector for Modifying Bacterial Genomes Using a CRISPR-Associated Transposon

CRISPR-associated transposons (CASTs) are RNA-guided mobile genetic elements that are widespread in bacterial genomes. Here, we describe the UltraCAST, a suicide vector with the Vibrio cholerae Type I-F CAST system and Golden Gate assembly sites with fluorescent protein gene dropouts for guide RNA and a mini-transposon cargo cloning. We show an example of UltraCAST genome editing by disrupting a gene in the chromosome of Serratia symbiotica CWBI-2.3 ^T , a culturable relative of aphid endosymbionts. The UltraCAST can be used to flexibly insert DNA into specific genomic sites and facilitates testing this genome editing platform in non-model bacterial species that lack genetic tools.