1. Efficient genome editing for Pseudomonas aeruginosa using CRISPR-Cas12a
- Author
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Yanyun Jing, Zhanglin Lin, Tingting Wang, Yanrui Ye, Huanhuan Li, Lan He, and Marco Pistolozzi
- Subjects
Gene Editing ,0301 basic medicine ,Trans-activating crRNA ,Cas9 ,General Medicine ,Bacterial genome size ,Computational biology ,Biology ,Genome engineering ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Bacterial Proteins ,Genome editing ,030220 oncology & carcinogenesis ,Pseudomonas aeruginosa ,Genetics ,Recombinase ,CRISPR ,CRISPR-Cas Systems ,Gene ,Genome, Bacterial ,Plasmids - Abstract
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 λRed 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.
- Published
- 2021
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