Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris.

Autor: Garay-Novillo JN; Departamento de Química Biológica Ranwel Caputto, CIQUIBIC-CONICET, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.; Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, Madrid, Spain., Ruiz-Masó JÁ; Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, Madrid, Spain., Del Solar G; Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, Madrid, Spain., Barra JL; Departamento de Química Biológica Ranwel Caputto, CIQUIBIC-CONICET, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
Jazyk: angličtina
Zdroj: Microbial biotechnology [Microb Biotechnol] 2024 Dec; Vol. 17 (12), pp. e70060.
DOI: 10.1111/1751-7915.70060
Abstrakt: In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity, and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.
(© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)
Databáze: MEDLINE
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