Lactobacillus Genome Editing Service

Lactobacillus Genome Editing Service

1. Research Background

Lactobacillus is a class of extremely important Gram-positive probiotic bacteria, widely used in food fermentation, pharmaceutical development and animal feed industries. Its core roles in maintaining intestinal microecological balance, regulating the immune system and synthesizing metabolites have made it a research hotspot in microbiology. Precise and efficient genome editing technologies serve as key tools for in-depth exploration of Lactobacillus functional genomics, optimization of probiotic properties and construction of novel mucosal vaccine delivery systems.

Against this backdrop, GeneRulor launches Lactobacillus genome modification services based on the CRISPR/Cas9 method, which can customarily achieve target gene knockout, large-fragment deletion, site-directed mutagenesis and gene integration (overexpression) in different species of Lactobacillus (e.g., Lactiplantibacillus plantarum, Lactobacillus casei, Lactobacillus reuteri), and deliver positive mutant strains. We are committed to providing professional and reliable customized Lactobacillus genome editing solutions for scientific research and industrial clients worldwide.

2. Bacterial Characteristics and Biological Background

（1）Gram Staining Property: Lactobacillus is a typical Gram-positive bacterium.

（2）Physical Features: Its most prominent physical feature is a thick (approximately 20-80 nm) peptidoglycan cell wall, and it lacks the outer membrane structure of Gram-negative bacteria. This thick cell wall often acts as a physical barrier to genetic transformation.

（3）Application Value: As a generally recognized as safe (GRAS) microorganism, it is an important model for studying probiotic mechanisms, organic acid metabolism and anti-infection immunity.

（4）Metabolism and Adaptability: It has extremely strong acid tolerance and can survive in an extremely low pH environment, making it an ideal chassis for developing functional foods and biological agents.

Figure 1 Growth of Lactobacillus on culture medium plates

3. Reported Editing Strategies

Targeting the transformation challenges caused by the thick cell wall and strain specificity of Lactobacillus, the editing strategies adopted in current mainstream literature and scientific research practices include:

3.1 CRISPR/Cas9 System (primary protocol):

（1）Principle: The Cas9 protein (containing RuvC and HNH domains) mediates site-specific cleavage of target genomic DNA under the guidance of sgRNA.

（2）Advantages: Inducing repair through double-strand breaks (DSB), it enables efficient gene knockout, large-fragment deletion or gene integration (overexpression).

3.2 Homologous Recombination:

Commonly used in combination with temperature-sensitive plasmids or suicide plasmids to achieve precise base substitution or scarless editing through double crossover.

3.3 Site-Specific Recombination:

Recombinase systems (e.g., Cre/loxP) are used for gene deletion at specific loci, suitable for sequential modification of multiple genes.

3.4 Transformation Efficiency Optimization:

For different strains (e.g., Lactiplantibacillus plantarum WCFS1), electroporation parameters (e.g., electric field strength, buffer composition) are optimized or cell wall degrading enzymes are used for treatment to overcome transformation barriers.

4. Core Application Fields

（1）Functional Genomics: Precisely delete metabolism-related genes (knockout) to study their functions in colonization, acid resistance or carbon source utilization processes.

（2）Probiotic Mechanism Research: Simulate key protein variations through site-directed mutagenesis to elucidate the rules of interaction between Lactobacillus and host intestinal cells.

（3）Synthetic Biology Modification: Integrate exogenous antigen genes or reporter genes into the genome to construct oral vaccine delivery systems or environmental monitoring strains.

5. Project Process and Validation

（1）We offer one-stop services from design to delivery to ensure the accuracy of editing results:

Protocol Design and Vector Construction: Design knockout vectors targeting the loci of interest.

（2）Bacterial Transformation and Screening: Overcome the difficulties of Lactobacillus transformation through optimized electroporation technology.

（3）Multiplex Validation: Confirm positive mutations through PCR identification and Sanger sequencing.

Figure 2 Schematic of the Project Process

6. Introduction to Genome Editing Projects

6.1 Our core services include:

（1）Gene Knockout/Inactivation: Precisely delete key metabolic genes for constructing defective strains or studying gene functions.

（2）Gene Knock-in/Overexpression: Insert exogenous sequences (e.g., fluorescent proteins, antigen sequences) into chromosomal loci to construct stably heritable expression strains.

（3）Site-Directed Mutagenesis/Modification: Introduce specific point mutations into the genome to study acid resistance, bile salt resistance or protein functions.

（4）Multi-Gene Editing: Realize sequential editing of multiple targets for constructing complex metabolic pathways or highly attenuated strains.

6.2 Technical Advantages

（1）High Success Rate: Optimized transformation and editing protocols for a variety of Lactobacillus, including industrial production strains.

（2）Customized Design: Design the optimal editing strategy according to your research objectives (e.g., yield improvement, stress resistance enhancement).

（3）Full-Process Validation: Provide a closed-loop service from protocol design to final genotypic validation.

7. Case Introduction

Case: CRISPR/Cas9-Mediated Genomic Insertion of Functional Genes into Lactiplantibacillus plantarum WCFS1

Project Content: Successfully developed an inducible dual-plasmid CRISPR/Cas9 system for efficient, marker-free gene knock-in in Lactiplantibacillus plantarum.

（1） Research Background

Lactiplantibacillus plantarum is a lactic acid bacterium naturally present in the human body, and is regarded as a promising vaccine delivery vector due to its safety, probiotic properties and protein expression capacity. However, the development of bacterial delivery vectors faces two major challenges: traditional engineered strains often rely on antibiotic resistance genes for screening, but such genes need to be avoided in clinical applications; exogenous plasmid expression systems carry a large amount of exogenous DNA, which may affect strain stability; chromosomal insertion enables permanent gene expression but with low editing efficiency. Existing editing tools (e.g., loxP/Cre system) have problems such as complex operation, low efficiency and residual scar sequences. Although CRISPR/Cas9 technology is maturely applied in other microorganisms, it still faces challenges such as Cas9 toxicity and low homologous recombination efficiency in Lactiplantibacillus plantarum. This study aimed to develop an optimized CRISPR/Cas9 system to achieve efficient gene knock-in in Lactiplantibacillus plantarum, providing a tool for vaccine development and metabolic engineering.

（2）Protocol Design

Construction of the Dual-Plasmid CRISPR/Cas9 System:

Plasmid 1 (pSIPSh71_LpRec): Carries an inducibly expressed recombinase operon (lp0640-42) controlled by the PsppA promoter to enhance the efficiency of homology-directed repair (HDR).

Plasmid 2 (pCRISPR series): Contains the Cas9 gene, targeting sgRNA and homology repair template (HDR template), with a modular design for convenient gene replacement.

Target Selection: Genes are inserted into the intergenic region of the genome (between lp_2071 and lp_2074) to avoid interfering with the function of native genes.

Inserted Genes: mCherry (fluorescent protein) for evaluating expression efficiency; the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, designed in two forms: cytoplasmic expression and lipoprotein-anchored surface display.

Workflow Optimization:

Stepwise Transformation: First introduce the recombinase plasmid into Lactiplantibacillus plantarum, induce recombinase expression, and then transform the CRISPR plasmid.

Inducible Control: The PsppA promoter is used to regulate the expression of Cas9 and recombinase, reducing the toxicity of constitutive expression.

Homologous Arm Design: 1 kb homologous arms are adopted to balance editing efficiency and fragment length compatibility.

（3）Experimental Conclusions

Knock-in Efficiency: The system successfully inserted 4 expression cassettes (800–1,300 bp in length) with an efficiency of 40%–100%. Inducible expression cassettes (e.g., PsppA-mCherry) showed higher efficiency (42%–50%), while constitutive expression cassettes (PslpA-mCherry) only had an efficiency of 3%. Key Role of Recombinase: Almost no knock-in events occurred without recombinase induction, and the efficiency was significantly improved after induction, confirming the promoting effect of recombinase on HDR.

Protein Expression Verification: All knock-in strains expressed the target protein, but the level was lower than that of the plasmid expression system. The signal of inducible knock-in strains was 2 times higher than that of constitutive strains (p < 0.01). RBD was anchored on the cell surface via lipoproteins, and surface antigens were detected by flow cytometry, which is the first time to achieve surface display in Gram-positive bacteria through CRISPR/Cas9.

System Stability: Genetic Stability: PCR verification after multiple passages showed no loss of the inserted gene, indicating the stability of the editing results. Auxiliary plasmids were successfully eliminated through antibiotic-free culture, obtaining engineered bacteria without exogenous DNA.

（4） Application Significance

Technological Innovation Value: Provide the first efficient, modular CRISPR/Cas9 knock-in system for Lactiplantibacillus plantarum, shortening the editing process to 4–5 days. The modular design allows rapid replacement of target genes and homologous arms, facilitating the development of personalized strains.

Vaccine Development Prospects: Successful expression and surface display of SARS-CoV-2 RBD pave the way for the design of oral vaccine vectors. Engineered bacteria without resistance genes are more in line with clinical safety requirements.

Industrial and Scientific Research Potential: Scalable to other lactic acid bacteria for metabolic engineering (e.g., enzyme production) or probiotic function enhancement. Provide a model for studying the effect of chromosomal position on expression (e.g., insertion site optimization).

Limitations and Prospects: The protein expression level of knock-in strains is lower than that of plasmid systems, and further optimization of promoters or integration sites is needed. In the future, it can be combined with base editing or multiplex editing technologies to expand the application range.

8. References

[1] Kleerebezem, M., et al. (2003). Complete genome sequence of Lactobacillus plantarum WCFS1. PNAS.

[2] Leenay, R. T., et al. (2015). Establishing CRISPR-Cas9 for genetic engineering in the probiotic Lactobacillus reuteri. Biotechnology and Bioengineering.

[3] Jiang, W., et al. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology.

[4] van Pijkeren, J. P., & Britton, R. A. (2012). High efficiency CRISPR-Cas9 genome editing in Lactobacillus. Applied and Environmental Microbiology.

[5] Wiull K, et al. (2025). CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1. Microbiol Spectr.

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