CRO Gene Editing Services

Corynebacterium glutamicum Gene Knock-in

Precise, CRISPR-mediated integration of target genes into the C. glutamicum genome, with marker-free integration available when scoped.

Custom guide RNA and donor template design for target locus
Homology-directed repair with verified integration junctions
Sequence-verified constructs and confirmed engineered strains, with glycerol stocks included as scoped.

Our service enables stable integration of heterologous genes or expression cassettes into the Corynebacterium glutamicum chromosome, facilitating long-term production without plasmid dependence. We handle strain evaluation, donor design, transformation, and rigorous clone screening. Deliverables include sequence-verified constructs and confirmed engineered strains as scoped, with data supporting downstream characterization and scale-up evaluation.

CRISPR/Cas9 or alternative systems as scoped
Marker-free integration options available
Screening by colony PCR and Sanger sequencing

GRAS Strain Host

Customizable Knock-in

Rigorous QC & Documentation

Our Approach

Strain Flexibility

Work with C. glutamicum ATCC 13032, ATCC 13869, or client-provided strains. Suitability for the intended use is assessed for the final strain and product.

Scalable Workflow

From single-gene knock-in to multi-locus integration. Screening depth and scope are defined per project.

Stability Assessment

QC includes serial passaging and re-sequencing to investigate genetic stability of the engineered strain.

What You Receive

Project Deliverables

All materials and data from your knock-in project, tailored to the agreed scope.

Strain & Samples

Sequence-verified engineered C. glutamicum strain (1 glycerol stock)
Parental control strain (glycerol stock)
Plasmid constructs (donor and CRISPR vectors) when requested
QC documentation including sequencing traces and PCR gel images

Data & Reports

Integration confirmation report with sequence alignments
Off-target analysis report (in silico + optional amplicon sequencing)
Project-scoped documentation and materials as agreed at kickoff.
Final certificate of analysis

Service Scope

Parameter	Typical project scope
Target Strain	C. glutamicum GRAS strains (ATCC 13032, ATCC 13869, or client-provided isolate)
Gene of Interest (GOI) Size	Scoped per project after consultation
Homology Arm Length	Scoped per project after consultation
Marker Integration	Marker-free or antibiotic marker (kanamycin resistance, chloramphenicol resistance) as selected
Clone Screening Depth	Typically 24–96 colonies; can be increased per project
Integration Locus	Intergenic site (e.g., cg1121-cg1122) or specific locus as requested

Workflow

How We Deliver Your Knock-in Strain

A streamlined, five-step process from design to validated strain.

Strain & Target Evaluation

We review your target gene sequence, intended integration locus, and strain background. Guide RNAs are designed for the site, and homology arm boundaries are defined based on genomic context.

Donor & CRISPR Construction

Donor template containing the gene of interest with homology arms is synthesized and cloned into a delivery vector. Guide RNA expression cassettes are assembled and verified by sequencing.

Transformation & Selection

C. glutamicum competent cells are prepared and co-transformed with the donor and CRISPR plasmids. Colonies are selected on appropriate antibiotics or marker recovery plates.

Clone Screening & Confirmation

Candidate clones are screened by colony PCR spanning both integration junctions. Positive clones are verified by Sanger sequencing to confirm the intended sequence and junctions; expression requires a separate readout.

Strain Purification & QC

Verified clones are streak-purified to single colonies. Glycerol stocks are prepared, and a final QC panel including junction PCR, sequencing, and optional stability passaging is completed.

Applications

Where Knock-in Strains Add Value

Stable gene integration supports a range of industrial and research uses.

Metabolic Pathway Engineering

Knock-in of key enzymes or transporters to evaluate production of lysine, glutamate, or other amino acids.

Overexpression of rate-limiting enzymes
Integration of heterologous pathways
Stable expression without plasmid loss

Recombinant Protein Expression

Integrate genes for industrial enzymes, therapeutic proteins, or biosensors into the C. glutamicum chromosome.

Inducible or constitutive promoters as needed
Secretion signal optimization available
Long-term expression stability

Cell Factory Construction

Combine multiple knock-ins for complex metabolic rewiring, including co-factor regeneration and byproduct elimination.

Multi-locus integration possible via marker recycling
Marker-free strains for successive rounds
Flux balance analysis support (optional)

FAQ

Frequently Asked Questions

Common concerns about our C. glutamicum knock-in service.

Insert size depends on the donor design and strain. The feasible insert size is evaluated during project design. Larger fragments may require case-by-case optimization; we evaluate during the design phase and propose the best strategy.
Yes. We offer marker-free integration using counter-selectable markers (e.g., sacB) or Cre-lox recombination. This allows for subsequent rounds of genome editing without retained antibiotic markers. Marker-free strains are available as an option scoped per project.
Integration is confirmed by colony PCR amplifying across both 5′ and 3′ junctions, followed by Sanger sequencing of the entire inserted region and flanking genomic DNA.
Project timelines are confirmed during scoping and cover design, construction, transformation, screening, and QC.

Scientific Literature

References

Wu M, Xu Y, Yang J, et al. Homing endonuclease I-SceI-mediated Corynebacterium glutamicum ATCC 13032 genome engineering. Applied microbiology and biotechnology. 2020;104(8):3597-3609. View on PubMed
Chang C, Nguyen MT, Ton-That H. Genetic Manipulation of Corynebacterium diphtheriae and Other Corynebacterium Species. Current protocols in microbiology. 2020;58(1):e111. View on PubMed
Peng F, Wang X, Sun Y, et al. Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system. Microbial cell factories. 2017;16(1):201. View on PubMed
Wang X, Yang S, Qian F, et al. RAGATH-Associated DNA Nuclease Assisted DNA Insertion in Corynebacterium glutamicum. ACS synthetic biology. 2025;14(5):1861-1867. View on PubMed

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