Genome Engineering of Streptococcus

1. Service Overview

The service focuses on the genome modification of Streptococcus, which involves manipulating the Streptococcus genome using advanced gene editing technologies to achieve specific biological functions or phenotypic changes. Core services based on homologous recombination (HR) gene editing technology include three main types:

(1) Markerless gene knockout, enabling efficient site-specific gene knockout via the HR system;

(2) Gene knock-in, realizing site-specific insertion of target genes into the Streptococcus genome;

(3) Gene overexpression, achieving overexpression of target genes at specific genomic loci.

The entire service cycle is 2–3 months, aiming to provide precise and efficient genomic engineering solutions for Streptococcus-related research and industrial applications.

2. Technical Principle

The core technical principle of this service relies on the homologous recombination (HR) gene editing technology, which is based on the endogenous DNA repair mechanism of organisms. This technology utilizes the homologous sequences inherent in the organism to guide the insertion of exogenous DNA fragments into the genome, thereby achieving precise editing of target genes. Through HR technology, a series of genomic operations can be performed on Streptococcus, including subcloning, insertion, deletion, replacement, and point mutation of target genes. For gene insertion or overexpression, the Tn5 transposase system is further integrated to accelerate the integration process of target genes, ensuring efficient and stable genomic modification.

3. Technical Flowchart

Fig.1 Homologous recombination allows for a variety of editing strategies targeting genes

4. Technical Features

The HR-based Streptococcus genome modification technology exhibits four prominent features:

(1) High editing efficiency: Under antibiotic selection conditions, the technology enables efficient knockout of target genes, significantly improving the success rate of genomic engineering;

(2) Rapid gene integration: For gene insertion or overexpression projects, the Tn5 transposase system is employed to achieve rapid integration of target genes, shortening the operation cycle;

(3) Simple operation: After constructing the HR editing plasmid, the plasmid can be introduced into Streptococcus cells via electroporation or chemical transformation, reducing technical barriers;

(4) Strong targeting ability: The technology relies on homologous sequence pairing to achieve precise editing of specific genomic loci, avoiding off-target effects and ensuring the accuracy of modification.

5. Application Scenarios

The technology is widely applied in three major fields:

(1) Medical field: In dental caries research, genetically engineered Streptococcus mutans is developed—for example, constructing lactate dehydrogenase (ldh)-deficient mutants and inactivating the gcrr gene. This modification reduces the acid-producing ability of the strain while enhancing its adhesion and colonization capacity, laying a foundation for the development of biological products for caries prevention;

(2) Industrial field: Genomic modification of antibiotic-producing Streptococcus strains to improve the yield and quality of antibiotics. Additionally, engineered Streptococcus can be designed to synthesize bioactive substances with medicinal value, such as peptide drugs and polysaccharide drugs;

(3) Basic research field: Constructing gene deletion or overexpression strains of Streptococcus through genomic modification to study gene functions, as well as the metabolic pathways and pathogenic mechanisms of Streptococcus, providing critical technical tools for in-depth understanding of Streptococcus biological characteristics.

6. Case Demonstration

GeneRulor successfully achieved gene knockout in Streptococcus anginosus using the HR method: via HR combined with CSP-1 induction, the erythromycin (Erm) resistance gene was inserted into the target gene in the S. anginosus genome, resulting in effective inactivation of the target gene.

Fig.2 Gene knockout in Streptococcus anginosus via homologous recombination method

7. Delivery Content and Standards

The delivered content of the service meets the following standards:

(1) PCR and DNA sequencing verification data of the target genomic locus, ensuring the accuracy of the modification;

(2) 2 glycerol stocks of the engineered Streptococcus strain, guaranteeing the preservation and usability of the strain;

(3) mRNA transcription level data (exclusively for gene overexpression projects), verifying the overexpression efficiency of the target gene;

(4) A comprehensive project final report, including detailed experimental procedures, results analysis, and technical summaries.

References

Zhu L, Li M, Yu G, et al. Investigation of choline-binding protein of CbpD in the pathogenesis of Streptococcus suis type 2.Front Vet Sci. 2024;11:1486347. Published 2024 Dec 3. doi:10.3389/fvets.2024.1486347