Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering.

Autor: Choi SY; Program of Biopharmaceutical Science and Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University, Gyeonggi, 13488, Republic of Korea., Romero-Calle DX; Program of Biopharmaceutical Science and Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University, Gyeonggi, 13488, Republic of Korea., Cho HG; Program of Biopharmaceutical Science and Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University, Gyeonggi, 13488, Republic of Korea., Bae HW; Program of Biopharmaceutical Science and Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University, Gyeonggi, 13488, Republic of Korea., Cho YH; Program of Biopharmaceutical Science and Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University, Gyeonggi, 13488, Republic of Korea. youhee@cha.ac.kr.
Jazyk: angličtina
Zdroj: Journal of microbiology (Seoul, Korea) [J Microbiol] 2024 Jan; Vol. 62 (1), pp. 1-10. Date of Electronic Publication: 2024 Feb 01.
DOI: 10.1007/s12275-024-00107-2
Abstrakt: Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.
(© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)
Databáze: MEDLINE
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