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

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: CRISPR-transposase; CRISPR/Cas9; Cre/lox; food biotechnology; genome editing; homologous recombination; lactic acid bacteria; probiotic engineering; recombineering; transposon mutagenesis.

Figure 1

Schematic RecA-mediated recombination. RecA proteins help anneal ssDNA to the chromosome or plasmid, forming triple-stranded DNA complexes, after which ssDNA fragments integrate into dsDNA.

Figure 2

λ-Red-mediated integration of foreign DNA. Exo protein resects dsDNA, Bet protein binds and anneals the resulting ssDNA to the lagging strand of the replicative fork, and Gam protein inhibits host’s nucleases protecting the ssDNA. After annealing foreign ssDNA assimilates with the host’s genome or plasmid DNA resulting in gene disruption or integration depending on the DNA inserted.

Figure 3

Target gene integration into genomic DNA via Cre/loxP system. For more precision two different loxP sites are needed. During the final loxP excision target gene will be integrated into the genome half of the time and cells that had this swap occur can be further enriched for or screened in the absence of the selective marker.

Figure 4

Principle of CRISPR/Cas counter-selection. Plasmid with CRISPR/Cas contains homologous to the gDNA regions which flank target gene. If homologous recombination occurs, then target gene region on the gDNA mutates (either by removing target gene entirely or by mutating it otherwise) in which case Cas protein cannot cut gDNA and the cell survives. If recombination does not occur, then Cas protein simply cuts gDNA in the target region and the cell dies. Cas9 is shown as blue circle with black sgRNA inside. Red rectangle shows the recognition site of Cas9 on the gDNA. Green and yellow rectangles show the adjacent parts of gDNA that can recombine with the plasmid.

Figure 5

DNA repair after (1) CRISPR/Cas cut or (2) double nickase cut. (A) shows a homology-directed repair which can create precise knock-ins or knockouts depending on the donor DNA. (B) shows a non-homologous end joining by either a deletion or an insertion which can disrupt the target region and even the whole locus.

Figure 6

Efficient large DNA fragment insertion via a CRISPR-associated transposon. Using catalytically active Cas9 can lead to errors due to DSB creation at the recognition site, which is why it is beneficial to use either dCas9 or different Cas proteins (such as Cas12k or Cas6) to increase the success rate of transposition.

Figure 7

CRISPRi and CRIPSRa systems. (A) catalytically dead Cas protein is fused with the inhibitor (or transcription repressor; although in some cases repression is achieved via steric hindrances without inhibitor domain) and when is bound to the target region it cannot cleave it inhibits the transcription of the target gene. (B) catalytically dead Cas protein is fused with the transcription activator and when is bound to the target region it cannot cleave it activates the transcription of the target gene.