Efficient genome editing for Pseudomonas aeruginosa using CRISPR-Cas12a

作者：Lin, Zhanglin^*; Li, Huanhuan; He, Lan; Jing, Yanyun; Pistolozzi, Marco; Wang, Tingting^*; Ye, Yanrui^*

来源：Gene, 2021, 790: 145693.

DOI：10.1016/j.gene.2021.145693

• FnCas12a nuclease
• CRISPR
• Genome editing
• Large DNA deletion
• Pseudomonas aeruginosa

The CRISPR-Cas12a system has been demonstrated as an attractive tool for bacterial genome engineering. In particular, FnCas12a recognizes protospacer-adjacent motif (PAM) sites with medium or low GC content, which complements the Cas9-based systems. Here we explored Francisella novicida Cas12a (FnCas12a) for genome editing in Pseudomonas aeruginosa. By using a two-plasmid system expressing the constitutive FnCas12a nuclease, the inducible lambda Red recombinase, a CRISPR RNA (crRNA), we achieved gene deletion, insertion and replacement with high efficiency (in most cases > 75%), including the deletion of large DNA fragments up to 15 kb and the serial deletion of duplicate gene clusters. This work should provide a useful and complementary addition to the genome engineering toolbox for the study of P. aeruginosa biology and physiology.