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Review

Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions

by
Leonid A. Shaposhnikov
*,
Aleksei S. Rozanov
* and
Alexey E. Sazonov
Scientific Center of Genetics and Life Sciences, Sirius University of Science and Technology, Sirius 354340, Russia
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(15), 7483; https://doi.org/10.3390/ijms26157483 (registering DOI)
Submission received: 9 July 2025 / Revised: 25 July 2025 / Accepted: 31 July 2025 / Published: 2 August 2025
(This article belongs to the Special Issue Probiotics in Health and Disease)

Abstract

Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications.
Keywords: lactic acid bacteria; genome editing; recombineering; CRISPR/Cas9; CRISPR-transposase; homologous recombination; Cre/lox; transposon mutagenesis; probiotic engineering; food biotechnology lactic acid bacteria; genome editing; recombineering; CRISPR/Cas9; CRISPR-transposase; homologous recombination; Cre/lox; transposon mutagenesis; probiotic engineering; food biotechnology

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MDPI and ACS Style

Shaposhnikov, L.A.; Rozanov, A.S.; Sazonov, A.E. Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions. Int. J. Mol. Sci. 2025, 26, 7483. https://doi.org/10.3390/ijms26157483

AMA Style

Shaposhnikov LA, Rozanov AS, Sazonov AE. Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions. International Journal of Molecular Sciences. 2025; 26(15):7483. https://doi.org/10.3390/ijms26157483

Chicago/Turabian Style

Shaposhnikov, Leonid A., Aleksei S. Rozanov, and Alexey E. Sazonov. 2025. "Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions" International Journal of Molecular Sciences 26, no. 15: 7483. https://doi.org/10.3390/ijms26157483

APA Style

Shaposhnikov, L. A., Rozanov, A. S., & Sazonov, A. E. (2025). Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions. International Journal of Molecular Sciences, 26(15), 7483. https://doi.org/10.3390/ijms26157483

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