Table 2. Tools for chromosomal editing of probiotic LAB strains.

*Genome Editing Tools	Notes	LAB in which application was successful	Reference(s)
Homologous recombination using the pORI system	● Homologous recombination via a non-replicating plasmid ● Can be used for gene deletion and insertion of expression cassettes based on conditional replication of vector pORI19	Lc. lactis, L. acidophilus and L. gasseri	[111–113]
	Advantages ● Does not dependent on transformation efficiency ● Enables growth of engineered strains at preferred growth temperatures ● Enables efficient recovery of stable integrants ● Applicable to deleting any non-essential gene across a broad range of species ● Seamless genome editing
	Limitations ● Plasmid DNA and antibiotic markers remain integrated in the chromosome, this complicates applications ● Time consuming and laborious
pTRK system	● Site-specific chromosomal integrations and deletions	L. acidophilus, L. gasseri, L. casei, Lc. lactis, L. plantarum	[114,115]
	Advantages ● Host temperature range that includes thermophilic lactobacilli ● Independent of transformation efficiency ● Can be used for marker-less gene replacement by using upp as counter-selectable marker for positive selection of double recombinants
	Limitations ● The stability of the insertional mutations after single-crossover HR requires maintenance of antibiotic selection ● The same selection marker cannot be used to introduce multiple mutations into a strain ● Insertional inactivation of a specific target within an operon may have polar effects on downstream region
Cre-lox system	Can be used for deletions and insertions	L. plantarum, L. casei, L. lactis	[69,70,116]
	Advantages ● Allows the removal of selectable marker(s) upon marker selection of deletion variant
	Limitations ● The presence of multiple loxP sites recognisable by Cre might lead to genome instability
ssDNA recombineering	● Homologous recombination of single- stranded linear DNA utilising λ-Red enzymes Gam, Exo, and Bet ● Targeted chromosomal mutation ● When assisted by RecT-mediated recombination, mutagenesis efficiencies of 0.4 to 19% can be achieved	L. reuteri, Lc. lactis, L. plantarum, L. gasseri	[19]
	Advantages ● Site specific ● Not hyper mutagenic ● Efficient for subtle genome modifications ● Allows selection of mutants without antibiotic marker selection
	Limitations ● Recombineering efficiency is dependent on expression of RecT homologs ● Inefficient for large chromosomal modifications (>1 kb) ● Selection of the desired mutations can be laborious and time consuming
dsDNA recombineering	● Recombinase-mediated deletions and insertions	L. plantarum, L. casei, L. paracasei	[117,118]
	Advantages ● Enables manipulation of large genomic regions ● Easy screening of mutants ● High efficiencies for both deletion and insertion
	Limitations ● Efficiency is dependent on specific interactions between recombinases and host-encoded proteins ● Antibiotic selection is required for higher efficiency of genome editing ● Removal of antibiotic markers employs Cre/loxP leaving a lox scar on the genome
CRISPR-Cas9	Precise genome editing using CRISPR/Cas9	Lactobacillus crispatus, L. plantarum, L. reuteri	[101,119]
	Advantages ● Enables programmable, precise genome editing ● High efficiency (up to 100%) for small deletions ● Marker free selection ● Have multiplexing potential, several deletions or genome modifications can be performed concurrently
	Limitations ● Transformation independent ● Limited to genetic sites with protospacer adjacent motif (PAM) motifs present ● Can introduce lethal double-strand breaks in off-target sites
ssDNA recombineering + CRISPR-Cas9	ssDNA recombineering combined with CRISPR/Cas9 for targeted chromosomal mutations	L. reuteri, Lc. lactis, L. gasseri, L. plantarum	[95,120,121]
	Advantages ● Allows genome editing without relying on restriction enzymes or antibiotic markers ● Introducing a CRISPR-Cas9 plasmid into ssDNA-recombineered bacteria can eliminate many of the unedited cells and improve the efficiency to >75% ● Time efficient (workflow can be completed within 72 h)
	Limitation ● Inefficient for large chromosomal modifications (>1 kb)
dsDNA recombineering + CRISPR-Cas9	Used to generate point mutations, deletions, insertions, and gene replacements	L. plantarum, L. brevis	[98,120,122]
	Advantages ● Effective, precise genome editing ● Seamless genome editing (sgRNA removes the loxP site)
	Limitations ● Lactobacilli respond to CRISPR-Cas9‐induced DSBs in a differently; therefore, the efficiency is difficult to predict ● A DSB or a nick may be ineffective in triggering HDR, resulting in cell death
CRISPR-Cas9D10A Nickase-assisted plasmid toolbox (pLCNICK)	Gene deletion and insertion using the Cas9D10A (nickase) ● Efficiency for deletions and insertions between 25 to 62% ● Correlation between deletion size and efficiency	L. casei, L. acidophilus, L. gasseri, and L. paracasei.	[99]
	Advantages ● Efficient, rapid, and precise tool for genome editing in L. casei ● Can circumvent DSB-induced lethality, probably due to variant repair pathways of nicks ● Marker free
	Limitations ● Inefficient for large deletions ● High fatality due to Cas9/sgRNA-induced DSBs
CRISPRi	Incorporation of dCas9 nuclease and sgRNA into the chromosome is required for genome editing	L. plantarum, Lc. lactis	[101,123,124]
	Advantages ● Repression of single or multiple target genes simultaneously ● dCas9 has an easily replaceable 20-nucleotide base-pairing region that can be programmed to target any gene of interest ● Enables easy down-regulation of any gene of interest ● Reversible effects ● Marker free ● Easier screening tool ● High potential for multiplexing ● Can be used for essential gene studies
	Limitations ● Silencing of non-target genes ● High levels of dCas9 expression coupled with off target binding of sgRNA can be toxic to cells ● CRISPRi system is active even without induction, this could affect essential genes and result in slower growth
Cre-loxP system	Site specific deletions, insertions, translocations, and inversions at specific sites	Lc. lactis, L. plantarum	[69,70,116]
	Advantages ● Can be used for simplified and programmable construction of large-scale chromosomal deletions (up to 39 kb) ● Very effective and precise due to the high affinity of the Cre recombinase for loxP sites ● Enables marker-less deletion, no need for counter selection ● Can be used for sequentially generating multiple deletions
	Limitations ● Very laborious and time consuming because it involves extensive selection and screening ● Cre/loxP carries the risk of creating unwanted effects at non target sites, and can be mutagenic
Selection/counter-selection marker system	Use of upp or mazF for chromosomal gene deletions, integrations, and deletions in LAB	L. plantarum 423, Enterococcus mundtii ST4SA, L. casei, Lc. lactis, L. acidophilus	[100,102,114]
	Advantages ● Efficient deletion or integration of genes at specific loci ● The upp counterselectable marker is recyclable ● Resulting in transgenic or mutant strains do not contain any selectable markers or residual plasmids ● Enables construction of stable double-crossover mutants
	Limitations ● The upp gene is involved in the nucleotide metabolic pathway of almost every organism ● 5-fluorouracil (5-FU) may be toxic, even in upp mutants; this complicates the use of counterselection for heterologous upp expression ● Identifying and optimising suitable counter-selection markers can be challenging and laborious.