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CRISPR/Cas utilizes base-pairing between complementary RNA and DNA to target specific sequences. This
[166]
makes it more accurate and efficient . Furthermore, CRISPR/Cas technology only requires the design of
guide RNAs (gRNAs) to target specific DNA sequences, making it simpler than designing the specific
[167]
proteins needed for ZFNs and TALENs . Once the designed gRNA-Cas complex creates a DNA double-
strand break (DSB) at the target site, the host cell can repair the break through either the non-homologous
end joining (NHEJ) or homology-directed repair (HDR) pathways . Recently, DSB-independent CRISPR/
[168]
Cas technologies have also been developed, including base editing (BE), prime editing (PE), and CRISPR-
associated transposases (CAST), which offer more precise methods for genome modification without
causing DSBs [169-171] .

Unlike plasmid-based overexpression systems, CRISPR/Cas gene editing allows for precise deletions,
additions, and replacements of a strain’s genome, offering a solution to the genetic instability associated
with plasmids and the contamination from antibiotic resistance genes. This approach is poised to become a
focal point in the development of engineered probiotics. We first examined the application of CRISPR/Cas
technology in two major genera of probiotics - Lactobacillus and Bifidobacterium. Our genomic analysis
revealed that CRISPR repeats were present in 59.7% of Lactobacillus genomes and 57% of Bifidobacterium
genomes [172,173] . Specific subtypes and sequences were identified, laying the groundwork for future gene
editing efforts. For example, Song et al. designed a plasmid, pLCNICK, which carries the Cas9D10A enzyme
and a sgRNA expression cassette, along with the homology arms of the target gene. This plasmid was
introduced into Lactobacillus casei via electrotransformation. Upon guiding Cas9D10A to induce a double-
stranded break at the target site, homologous recombination repair allowed for the replacement or
knockout of the target gene. The efficiency of this gene knockout ranged from 25% to 62% . Alternatively,
[174]
gene knockout can be achieved by obstructing RNA polymerase function with sgRNAs and partially
inactivated dCas9 (which cannot cleave DNA), thus creating a physical barrier to transcription .
[175]

Endogenous CRISPR/Cas systems, in contrast to exogenous ones, offer higher specificity and lower
cytotoxicity, which could make them more suitable for use in probiotics . Numerous endogenous
[176]
CRISPR/Cas systems are being developed for application in probiotics. As an example, researchers
reprogrammed the endogenous type I-G CRISPR-Cas system in Bifidobacterium animalis subsp. lactis to
identify naturally occurring large deletions and generate a 500-bp deletion in the tetW gene, thereby
[173]
eliminating tetracycline resistance . The translation of uridine phosphate ribose transferase in
Bifidobacterium breve FJSWX38M7 was successfully terminated by a single base substitution and the
insertion of three stop codons, using the endogenous Type I-C CRISPR-Cas system of
Bifidobacterium breve . The endogenous Type I-E CRISPR-Cas system was identified in Lactobacillus
[177]
crispatus NCK1350, with Cas3 as its signature protein. After identifying 5’-AAA-3’ as the PAM sequence,
the researchers designed the pTRK1183 plasmid containing the specific crRNA. This plasmid successfully
facilitated gene deletion (100% efficiency), stop codon insertion (36% efficiency), and single nucleotide
substitution (19% efficiency) in Lactobacillus crispatus NCK1350 . Although CRISPR/Cas-based gene
[176]
editing is still in its developmental phase, significant progress is being made, and several tools are under
development. Notably, there are few reports on the direct introduction of large heterologous protein
segments into probiotic genomes. A recent study, however, demonstrated successful expression of the HIV-
1 membrane-proximal external region (MPER) on the surface of EcN using CRISPR/Cas9. This
breakthrough suggests the potential of engineered probiotics to enhance mucosal immune responses . As
[178]
research into CRISPR/Cas continues, it will deepen our understanding of the genetic characteristics of
probiotics and open new avenues for their application. Table 2 summarizes the application of CRISPR-Cas-
based gene editing technology in some probiotics [Table 2].