Yeast Gene Knock-in Service

The Yeast Gene Knock-in Service provides precise and stable insertion of desired DNA sequences—ranging from single genes to entire biosynthetic pathways—directly into the yeast chromosome. Yeast (such as Saccharomyces cerevisiae and Pichia pastoris) is an ideal host for gene knock-in due to its robust native Homologous Recombination (HR) mechanism, which allows for efficient, targeted integration.

CD Biosynsis utilizes advanced techniques, often integrating CRISPR-Cas9 technology to significantly enhance the efficiency of the Double-Strand Break (DSB) required for targeted integration. This service is crucial for metabolic engineering projects that require high copy number expression and superior genetic stability, eliminating the risk of plasmid loss and ensuring consistent performance in industrial-scale fermentation. We deliver strains with verified, scarless knock-in edits, ready for immediate application.

Highlights

Key advantages of our Yeast Gene Knock-in Service:

• Enhanced Stability: Chromosomal integration ensures the inserted gene is passed down reliably, preventing the genetic drift associated with plasmids.
• High Integration Efficiency: Leveraging natural HR combined with Cas9 technology achieves high rates of targeted, template-driven integration.
• Pathway Assembly: Ideal for inserting multi-gene cassettes or entire biosynthetic pathways into the genome at pre-determined, stable loci.
• Precise Expression Tuning: Allows for insertion under native or optimized chromosomal promoters, ensuring predictable and stable gene expression levels.

Insertion Applications

Applications benefiting from high-precision yeast gene insertion:

Biosynthetic Pathway Integration

Stable, permanent insertion of large gene clusters for the production of non-native chemicals or compounds.

High-Copy Integration (HCI)

Targeted insertion into multiple chromosomal loci (e.g., rDNA regions) to achieve high copy numbers for high-yield protein expression.

Reporter and Tagging

Scarless insertion of fluorescent reporters (e.g., GFP) or affinity tags (e.g., His-tag) at the N- or C-terminus of native genes.

Allele Replacement and Correction

Precise replacement of a native gene or defective allele with a desired variant for functional analysis or correction.

Key Features & Stability

Technical features ensuring successful, stable yeast gene knock-in:

Optimized Donor Design

Creation of linearized DNA donors with homology arms tailored for high HR efficiency and scarless insertion.

CRISPR/HR Synergy

Combination of Cas9-induced DSB with HR to achieve significantly higher integration rates than traditional marker-based methods.

Large Construct Integration

Proven methodology for the stable insertion of large gene clusters up to 10 kb, ideal for pathway reconstruction.

Multiplex Integration

Ability to integrate multiple genes simultaneously into different chromosomal sites for efficient pathway tuning.

Verified Stability

Final strains are cured of transient editing tools, ensuring the long-term genetic stability required for fermentation.

Workflow

Our systematic workflow for high-precision yeast gene knock-in:

• Design and Template Synthesis: Design gRNAs and synthesize the linear DNA donor template containing the gene of interest and homology arms.
• Transformation and Integration: Co-transform the yeast host with the Cas9/gRNA expression system and the linear donor DNA.
• Selection and Clone Isolation: Select for successful integrants using a transient or selectable marker system.
• Verification: Perform molecular verification, including junction PCR and full sequencing of the edited locus, to confirm precise integration.
• Curing and Final Strain Delivery: Remove the Cas9 editing plasmid (curing) to ensure genetic stability, and deliver the final verified strain.

We provide essential assurance for high-quality yeast gene knock-in outcomes:

• Guaranteed Integration: Commitment to deliver a verified yeast clone with the target DNA sequence successfully integrated into the chromosome.
• Sequence Fidelity: Guaranteed correct sequence of the inserted cassette and verified seamless insertion at the intended locus.
• Long-Term Stability: Assurance that the final strain is highly stable and ready for scale-up due to chromosomal integration and removal of transient tools.
• Custom Locus Targeting: Expertise in targeting specific, non-essential, and high-expression loci within various yeast genomes.