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Table 2. CRISPR-Cas system in some probiotics
Probiotics Gene editing tools Application Ref.
Lactococcus lactis Cas9 protein, sgRNA, Constructed pMG-Cas9-ldh recombinant plasmid to knock out lactate dehydrogenase [179]
NZ9000 Red/ET recombinase gene; developed a food-grade gene editing system to avoid the introduction of
system, pMG36e plasmid antibiotics and exogenous genes
Lactobacillus Cas9, sgRNA, HMME, ZIF-8 Self-driving CRISPR/Cas9 nanosystems can reprogram TIME through multiple [180]
rhamnosus GG pathways
Lactobacillus Cas9, sgRNA, pNcas Obtained a thermotolerant strain NCBIO01-M2-ldhL1-HT capable of efficiently [181]
paracasei NCBIO01- producing L-lactic acid at 45 ℃
M2
Lactobacillus reuteri Cas9, tracrRNA, RecT, CRISPR-Cas9-assisted recombination techniques can facilitate targeted codon [182]
ATCC PTA 6475 crRNA, pVPL3017, saturation mutations. Additionally, CRISPR-Cas9-based selection enables the
pVPL3004 identification of mutations that occur at low frequencies, such as those resulting from
oligonucleotide-mediated chromosomal deletions
Bifidobacterium CRISPR-CBE A large segment deletion was produced in Bifidobacterium animalis subsp. lactis using [173]
animalis subsp. lactis the endogenous CRISPR-Cas system; the CRISPR-cytosine base editor was able to
introduce SNPs in B. lactis
EcN Cas3, crRNA, λ-RED Engineered EcN is effective in removing multiple ARGs under in vitro and in vivo [183]
recombination system, conditions
cascade protein
EcN Cas9, sgRNA, pCas9-KT Removal of two cryptic plasmids, pMUT1 and pMUT2, from EcN reduces metabolic [184]
burden; used for GABA production in an antibiotic-free system
EcN CRISPR-Cas9 plasmid, pDA, For targeted delivery of the CRISPR-Cas9 system to deep-seated tumors for [185]
ROS-responsive linker photothermal sensitization immunotherapy, liposomes (Lipo-P) loaded with CRISPR-
Cas9 plasmid were used to reduce the thermotolerance of tumor cells through gene
editing

HMME: Hematoporphyrin monomethyl ether; TIME: tumor immunosuppressive microenvironment; CRISPR-CBE: CRISPR-Cas9 gene editing
system, base editor; SNPs: single-nucleotide polymorphisms; EcN: Escherichia coli Nissle 1917; ARGs: antibiotic resistance genes; GABA: γ-
aminobutyric acid; pDA: polydopamine; ROS: reactive oxygen species.

Advances in artificial intelligence (AI) are expected to significantly impact research in the field of
engineered bacteria, particularly in the areas of editing, screening, and optimizing engineered strains. For
instance, the development of the GEDpm-cg platform offers an efficient, user-friendly, and flexible tool for
genome editing in C. glutamicum, which is anticipated to enable large-scale mutation analysis through
robot- and software-assisted systems . This platform aims to improve the understanding and engineering
[186]
of cellular metabolism. Moreover, structural biology plays a key role in the optimization of gene editing
tools. In particular, understanding the high-resolution structure of nucleases such as Cas9 is crucial for
elucidating how these enzymes recognize and cleave DNA. By analyzing the structure of Cas9,
modifications can be made to enhance its specificity and reduce off-target effects.

CONCLUSION
Natural probiotics have demonstrated beneficial properties such as improving intestinal microbiota,
regulating metabolism, and exhibiting antitumor effects. However, nonspecific probiotic supplementation
and FMT may lead to variable efficacy and potential safety concerns, making the development of engineered
bacteria for more targeted treatments a more sensible approach. Leveraging synthetic biology, researchers
are genetically engineering these probiotics to carry out specific functions beneficial to human health.
Engineered bacteria show promising potential for treating conditions such as IBD, metabolic disorders,
neurodegenerative diseases, and cancer. Despite successful results in animal models, clinical trials are
limited, with few products like SYNB1618 demonstrating safety and tolerability . None have yet been
[76]
approved for market use. Advancements in gene editing tools enable the design of complex genetic circuits
to finely tune the biological capabilities of engineered bacteria. This includes enhancing their targeting
[188]
[187]
specificity and optimizing the secretion of desired products . Despite their therapeutic promise,
challenges remain, such as improving safety profiles, refining clinical trial methodologies, and advancing