Bacteroides Genome Editing Service

Bacteroides Genome Editing Service

1.Research Background

Bacteroides is the most abundant and representative obligate anaerobic Gram-negative bacterium in the human gut microbiota. It plays a crucial role in polysaccharide metabolism, regulation of the host immune system and colonization resistance. With the in-depth research on the "microbiota-gut-brain axis" and symbiotic mechanisms, precise and efficient genome editing technologies have become core tools for elucidating Bacteroides functional genes, developing engineered probiotics and therapeutic strategies.

Against this backdrop, GeneRulor launches CRISPR/Cas and suicide plasmid homologous recombination solutions for Bacteroides, which can customarily achieve target gene knockout, site-directed mutagenesis and gene integration (overexpression) in different species of Bacteroides (e.g., B. thetaiotaomicron, B. fragilis), and deliver verified positive mutant strains. We are committed to providing professional and reliable Bacteroides genome modification solutions for scientific research clients worldwide.

2. Bacterial Characteristics and Biological Background

（1）Gram Staining Property: Bacteroides is a typical Gram-negative bacterium.

（2）Physical Features: It is an obligate anaerobe, non-sporulating, and has a complex capsular polysaccharide structure. Its outer membrane contains a unique lipopolysaccharide (LPS), which is different from that of common intestinal pathogenic bacteria.

（3）Clinical and Ecological Significance: Bacteroides are the major carbohydrate degraders in the gut. Although most are commensal bacteria, some species such as B. fragilis also have important clinical significance in intra-abdominal infections.

（4）Metabolism and Applications: It has a strong ability to utilize polysaccharides (mediated by PULs gene clusters), making it an ideal model for studying intestinal symbiotic mechanisms, complex carbon source metabolism and host-microbe interactions.

Figure 1 Growth of Bacteroides on anaerobic culture medium plates

3. Reported Editing Strategies

Targeting the challenges of Bacteroides being obligate anaerobes and having low conventional transformation efficiency, the mainstream strategies adopted in current literature include:

3.1 CRISPR/Cas System (e.g., Cas9 or Cas12a):

（1）Principle: Cas proteins are guided by sgRNAs to precisely cleave the target genome.

（2）Advantages: As a powerful negative selection tool, it greatly improves the efficiency of scarless editing, especially suitable for large-fragment deletion or gene integration.

3.2 Counter-selection System:

Suicide plasmids containing the tdk gene are commonly used in combination with 5-fluorodeoxyuridine (FUdR) for negative selection to achieve precise chromosomal replacement.

3.3 Conjugation:

Aiming at the unstable electroporation efficiency of Bacteroides, Escherichia coli (e.g., S17-1) is usually used as the donor bacterium to efficiently deliver editing plasmids into recipient Bacteroides via conjugation.

3.4 Transformation Efficiency Optimization:

For different Bacteroides species, optimize conjugation time, donor-recipient ratio and medium components to overcome the entry barrier caused by the thick capsule.

4. Core Application Fields

（1）Functional Genomics: Precisely delete polysaccharide utilization loci (PULs) to study their competitive advantages in the complex intestinal environment.

（2）Host-Microbe Interactions: Elucidate the regulatory mechanisms of Bacteroides on the intestinal immune system by knocking out virulence factors or surface polysaccharide synthesis genes.

（3）Synthetic Biology Chassis Construction: Insert therapeutic protein expression cassettes into genomic safe harbors to develop novel "live bacterial drug" delivery systems.

（4）Metabolic Pathway Modification: Optimize the synthetic pathway of short-chain fatty acids (SCFAs) through gene overexpression or site-directed mutagenesis.

5. Project Process and Validation

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

（1）Protocol Design and Vector Construction: Design knockout plasmids targeting Bacteroides loci of interest.

（2）Bacterial Transformation and Screening: Overcome the difficulties of transforming obligate anaerobes and screen integrated clones.

（3）Secondary Recombination and Resolution: Induce secondary recombination through negative selection or CRISPR cleavage.

（4）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 specific metabolic or signaling pathway genes for studying gene functions.

（2）Gene Knock-in/Overexpression: Integrate reporter genes (e.g., fluorescent proteins) or functional genes into chromosomal loci.

（3）Scarless Editing: No resistance markers are introduced, supporting multiple rounds of iterative modification of the same strain.

（4）Multi-Gene Editing: Realize sequential or simultaneous editing of multiple metabolic targets to construct complex engineered bacteria.

6.2 Technical Advantages

（1）Rich Experience: Covering multiple mainstream species such as B. thetaiotaomicron (Bt), B. fragilis (Bf) and B. vulgatus (Bv).

（2）Customized Design: Design the optimal strategy according to research objectives (e.g., polysaccharide metabolism, intestinal colonization).

（3）Full-Process Validation: Provide detailed sequencing reports and genotyping results.

7. Case Introduction

Case: Development of CRISPR/Cas-Based Genome Editing Tools for Bacteroides, a Human Gut Commensal Bacterium

Project Content: Successfully developed a highly efficient and multifunctional CRISPR/Cas genome editing platform for gene editing of Bacteroides, the dominant commensal bacterium in the human gut.

（1） Research Background

Bacteroides is the most abundant genus in the human gut microbiome (accounting for 20-30% of the gut microbiota) and plays a key role in maintaining intestinal health, metabolic regulation and immune balance. Its functional abnormalities are closely related to obesity, inflammatory bowel disease, colorectal cancer and other diseases. Although Bacteroides is an ideal model for studying gut microbial functions, its genetic manipulation tools still have limitations: methods such as NBU transposon or allelic exchange have low editing efficiency, rely on selectable markers, and cannot achieve marker-free editing; CRISPR/Cas technology is widely used in other bacteria, but optimized tools for strictly anaerobic Bacteroides species are still incomplete; there is an urgent need to develop efficient editing tools to elucidate the gene functions of Bacteroides (e.g., polysaccharide utilization loci PULs), metabolic pathways and their roles in the intestinal ecosystem. This study aimed to fill this gap and achieve precise, marker-free genome editing of Bacteroides by constructing a CRISPR/Cas system.

（2）Protocol Design

The research team designed an all-in-one plasmid system integrating CRISPR/Cas components, homology repair templates and host-adaptive elements. The core design includes:

CRISPR System Selection: Three Cas proteins were tested: FnCas12a (recognizing T-rich PAM), SpRY (nearly PAMless), and SpCas9 (classic system) to evaluate their editing efficiency. An inducible promoter (e.g., aTc-inducible P1TDPGH023) was used to control Cas expression to avoid the toxicity of constitutive expression.

Plasmid Construction and Delivery: A Bacteroides-E. coli shuttle plasmid was constructed and introduced into Bacteroides strains via conjugation. sgRNAs targeting specific genes (e.g., Bt1754) were designed, and homologous arms were introduced to mediate repair.

Workflow Optimization: Including five steps: plasmid construction, conjugation, Cas inducible expression, mutant screening and plasmid curing to ensure efficient editing.

Key Experiments: Promoter activity was evaluated by a luciferase reporter system (the inducible P1TDPGH023 had better activity than constitutive promoters). Species-specific sgRNAs were designed for different Bacteroides species (e.g., B. thetaiotaomicron, B. fragilis).

（3） Experimental Conclusions

Editing Efficiency and Optimization: The FnCas12a system had the highest efficiency, with a gene deletion efficiency of 60% in B. thetaiotaomicron (40% for SpRY and SpCas9), and the inducible promoter improved efficiency by more than 10 times compared with the constitutive one. In terms of large-fragment deletion capability, a PUL gene cluster as long as 50 kb (e.g., RGI-PUL) was successfully deleted, and the deletion efficiency of small fragments (5-10 kb) was nearly 100%.

Multifunctionality Verification: The gfp reporter gene was successfully inserted into the genome with an efficiency of over 80%, achieving fluorescent labeling. Cross-species application was realized, and the system was effective in B. fragilis, B. ovatus, B. uniformis and B. vulgatus with an editing efficiency of 60-100%. After deleting the metabolic gene (e.g., Bt1754), the growth of the mutant was inhibited in fructose medium, confirming the gene function. Plasmid curing experiments showed that the system supported multiple rounds of editing and avoided the residual of resistance genes.

（4） Application Significance

Scientific Research Value: Provide precise tools for studying the PUL metabolic network and host-microbe interactions of Bacteroides, such as elucidating the role of polysaccharide degradation pathways in the intestinal ecosystem. Support multiple gene editing and accelerate functional genomics research.

Biotechnology Applications: Strains can be engineered to enhance colonization ability and deliver therapeutic molecules (e.g., anti-inflammatory factors) for the treatment of metabolic diseases. It lays a foundation for gut microbiome engineering, design of synthetic microbial communities and regulation of the intestinal environment.

Technical Advantages and Prospects: High efficiency with the editing cycle shortened to 2-3 weeks, far exceeding traditional methods. Universal applicability, the tool is adapted to multiple Bacteroides species and promotes the standardization of gut microbiota research. In the future, combining with base editing, transcriptional regulation and other technologies can further expand the precise manipulation of gut microbes.

8. References

[1] Wexler, H. M. (2007). Bacteroides: the good, the bad, and the nitty-gritty. Clinical Microbiology Reviews.

[2] Mimee, M., et al. (2015). Programming a human commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to stimuli in the murine gut. Cell Systems.

[3] Hecht, A. L., et al. (2016). Composition and function of the human gut microbiome and its impact on health. Nature.

[4] Lim, B., et al. (2017). Engineered Bacteroides for the delivery of therapeutic proteins to the gut. Nature Communications.

[5] Zheng L, et al. (2022). CRISPR/Cas-Based Genome Editing for Human Gut Commensal Bacteroides Species. ACS Synth Biol.

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