RNA-Guided DNA Transposition in <i>Corynebacterium glutamicum</i> and <i>Bacillus subtilis</i>
Siqi Yang, Siqi Yang, Jiao Zhu, Xiaojie Zhou, Jiao Zhang, Qi Li, Furong Bian, Jidong Zhu, Ting Yan, Xiaoyu Wang, Ying Zhang, Junjie Yang, Yu Jiang, Sheng Yang, Sheng Yang
Abstract
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.