ArticleMethods in molecular biology (Clifton, N.J.)2022
Genome Editing with Cas9 in Lactobacilli.
Article in Methods in molecular biology (Clifton, N.J.), 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed.
- An optimized CRISPR/Cas9-based genome editing platform enhances bacteriocin production in Lactiplantibacillus plantarum.Journal of biological engineering · 2026Article
- Characterization of agr-like Loci in Lactiplantibacillus plantarum and L. paraplantarum and Their Role in Quorum Sensing and Virulence Inhibition of Staphylococcus aureus.Probiotics and antimicrobial proteins · 2025Article
- Development and Applications of CRISPR/Cas9-Based Genome Editing inInternational journal of molecular sciences · 2022Review
- CRISPR/Cas tools for enhancing the biopreservation ability of lactic acid bacteria in aquatic products.Frontiers in bioengineering and biotechnology · 2022Review
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Authors and funding
2 authors.
Funding
Abstract
The bacterial genus Lactobacillus comprises a vast range of strains with varying metabolic and probiotic traits, with genome editing representing an essential tool to probe genotype-phenotype relationships and enhance their beneficial properties. Currently, one of the most effective means of genome editing in bacteria couples low-efficiency recombineering with high-efficiency counterselection by nucleases from CRISPR-Cas systems. In lactobacilli, several CRISPR-based genome editing methods exist that have shown varying success in different strains. Here, we detail a fast and simple approach using two shuttle vectors encoding a recombineering template as well as the Streptococcus pyogenes Cas9, a trans-activating RNA, and a CRISPR array. We provide a step-by-step procedure for cloning the shuttle vectors, sequentially transforming the vectors into lactobacilli, screening for the desired edit, and finally clearing the shuttle vectors from the mutant strain. As CRISPR-based genome editing in bacteria can fail for various reasons, we also lay out instructions for probing mechanisms of escape. Finally, we include practical notes along the way to facilitate each stage of genome editing, and we illustrate the technique using a representative edit in a strain of Lactobacillus plantarum. Overall, this method should serve as a complete guide to performing genome editing in lactobacilli.
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Registered trials
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