Genome Engineering of   Rhizobium

1. Research Background

Genome Engineering of Rhizobium refers to the manipulation of its genome through gene editing technologies to achieve specific biological functions or phenotypic changes, mainly including the following application studies:

（1）Improving Nitrogen Fixation Efficiency: Rhizobium itself can form a symbiotic relationship with leguminous plants for nitrogen fixation, but the nitrogen-fixing capacity of natural strains may have certain limitations. Through engineering modification, the expression and regulation of nitrogen fixation-related genes can be optimized, and the activity of nitrogenase can be enhanced, enabling rhizobium to provide more available nitrogen for plants, reduce the application of chemical nitrogen fertilizers, and promote better plant growth.

（2）Enhancing Environmental Adaptability: After modification, it can survive, colonize and function under a wider range of soil environmental conditions, such as adapting to different pH values, temperatures, humidity, and soil microbial community compositions, which is conducive to its successful symbiosis with leguminous plants in farmland and other environments in different regions.

（3）Expanding Host Range: Natural rhizobium can often only form symbiotic relationships with specific types of leguminous plants. Modified engineering strains are expected to break this limitation, enabling them to symbiose, nodulate and fix nitrogen with more types of leguminous plants, thereby increasing the types of beneficial plants and improving the efficiency of nitrogen cycle utilization in the ecosystem.

2. Application of Gene Editing Technologies in Rhizobium

Advanced gene editing tools such as CRISPR-Cas and Tn5 transposase systems are used to precisely edit the genome of rhizobium. For example, knocking out negative regulatory genes that affect nitrogen fixation efficiency or inserting exogenous related genes can achieve targeted modification of its genetic information and obtain the desired excellent traits.

Figure 1: Schematic Diagram of CRISPR/Cas9 Cleavage and Tn5 Transposition

3. Service Types

（1）Marker-Free Gene Knockout: Efficient site-specific knockout is achieved using the CRISPR-Cas9 gene editing system, with the elimination of exogenous plasmids.

（2）Scarless Gene Point Mutation: The efficient site-specific cleavage of the CRISPR-Cas9 system is utilized to achieve site-specific genome mutation.

（3）Scarless Gene Knock-In: Site-specific genome knock-in is achieved using the CRISPR-Cas9 or Tn5 gene editing system.

（4）Gene Overexpression: Gene overexpression at specific genomic loci is achieved using the CRISPR-Cas9 or Tn5 gene editing system.

（5）Large-Segment Gene Deletion: Combined with the Red/ET recombination system and the CRISPR-Cas9 gene editing system, large-segment gene deletion in specific genomic regions is achieved.

4. Technical Advantages

（1）By combining traditional λRed homologous recombination technology with the advanced CRISPR-Cas9 scarless editing system, the homologous repair mechanism after efficient cleavage cooperates with the effect of simultaneous multi-site editing, and the efficiency of single-site gene editing can reach more than 90%;

（2）Rapid Gene Integration: The Tn5 transposase system enables the rapid insertion or overexpression of target genes;

（3）Scarless Editing: No resistance or recombination sites are left after target site editing, and exogenous plasmids are eliminated simultaneously;

（4）Simple Operation: After constructing the CRISPR editing plasmid, it is introduced into the target bacteria by electroporation for gene editing;

（5）Strong Targeting: CRISPR technology has the advantages of strong targeting and low off-target rate, enabling precise editing of target sites.

5. Project Cycle

2-3 months

6. Delivery Standards

（1）PCR and DNA sequencing identification data of target sites

（2）2 glycerol tubes of engineering strains

（3）mRNA transcription level data — Overexpression

（4）Project conclusion report

7. Success Case: Fluorescent Labeling of Bradyrhizobium Based on Tn5 Transposase System

Transposase is an enzyme that plays a key role in the transfer process of transposons (movable DNA sequences). It can realize efficient random insertion of exogenous genes, making it suitable for the overexpression and fluorescent labeling of target genes. By constructing a fusion of the red fluorescent protein mScarlet3 and resistance genes, then preparing the Tn5 Transposome RNP Complex, which was introduced into Escherichia coli and Bradyrhizobium by electroporation, fluorescent labeling was successfully achieved.

Figure 2: Fluorescent Labeling Results (Escherichia coli and Bradyrhizobium)

8. References

[1] Oldroyd, G.E.D. & Dixon, R. Biotechnological solutions to the nitrogen problem. Current Opinion in Biotechnology 26, 19–24 (2014). DOI: 10.1016/j.copbio.2013.08.006.

[2] Wang, L. et al. Genetic engineering of nitrogen-fixing bacteria: recent advances and future prospects. Biotechnology Advances 40, 107546 (2020). DOI: 10.1016/j.biotechadv.2019.107546.

[3] Mus, F. et al. Symbiotic nitrogen fixation and the challenges to its extension to nonlegumes. Applied and Environmental Microbiology 82, 3698–3710 (2016). DOI: 10.1128/AEM.01055-16.

[4] Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products (λ-Red recombineering). PNAS 97, 6640–6645 (2000). DOI: 10.1073/pnas.120163297.

[5] Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31, 233–239 (2013). DOI: 10.1038/nbt.2508.