Recently discovered CRISPR-associated transposases (CASTs) have been implemented as useful multiplexed genome editing tools, albeit only in a small group of bacteria. We demonstrated that the type I-F CAST from Vibrio cholerae could induce RNA-guided transposition in Bacillus subtilis and Corynebacterium glutamicum with efficiencies of 0.00018% and 0.027%, respectively. Lowering the culturing temperature to 16 °C in rich media increased the insertion efficiency to 3.64% in B. subtilis. By adding a positive selection against spectinomycin in the cargo DNA, up to 9 kb of the DNA fragment could be integrated at the target site with a 13.4% efficiency in C. glutamicum, which was difficult using the conventional approach. The successful implementation of CAST in these two academically important and industrial-relevant Firmicutes shows its great potential in a wide variety of bacteria and could be further optimized for multiplexed genome editing.
Keywords: Bacillus subtilis; CRISPR-associated transposases (CASTs); Corynebacterium glutamicum; large fragment insertion.