CD Biosynsis offers high-precision Saccharomyces cerevisiae Gene Knock-in Services, enabling the stable and accurate integration of large, heterologous DNA fragments into the yeast genome. S. cerevisiae (baker's yeast) is a widely used eukaryotic production host for synthesizing complex molecules, and its strength lies in its exceptional Homology-Directed Repair (HDR) efficiency. Our services leverage the CRISPR-Cas9 system to create targeted double-strand breaks (DSBs), dramatically increasing the rate of precise gene insertion (knock-in) at chosen genomic safe harbor sites. This capability is vital for installing multi-gene biosynthetic pathways, tagging endogenous proteins, and creating stable production strains without relying on unstable plasmids, ensuring high expression levels and reliable long-term performance.
Get a QuoteThe insertion of large DNA sequences, such as entire biosynthetic pathways, must be stable and highly expressed. Unlike traditional methods, our CRISPR-Cas9 platform uses the yeast's efficient HDR machinery to integrate the new DNA (donor template) precisely at the desired chromosomal location. This avoids random insertion and plasmid instability issues. We specialize in multi-site integration, allowing us to simultaneously insert multiple genes or pathways into different safe harbor sites, achieving higher product titers and managing the metabolic load effectively.
Safe Harbor Targeting
Identification of optimal genomic safe harbor loci (e.g., non-coding regions) for pathway integration, ensuring stable expression without disrupting essential native genes.
Large DNA Donor Design
Construction of long DNA templates (linear or circular) up to 50 kb, containing the pathway and optimized homology arms for high HDR efficiency.
Multi-Site/Multi-Copy Insertion
Strategies for simultaneous insertion of multiple gene copies or different pathways at distinct genomic loci to boost product yield or manage metabolic burden.
Cas9 and gRNA Delivery
Optimized delivery of the Cas9 expression vector and guide RNA (gRNA) plasmid/cassette into the S. cerevisiae host using high-efficiency transformation methods.
Marker Recycling System
Implementation of selection marker recycling (e.g., Cre-LoxP or auxotrophic restoration) to enable sequential knock-ins without accumulating drug resistance genes.
Promoter and Terminator Stacks
Design of optimized, standardized expression units (e.g., promoter-gene-terminator) to ensure high and stable expression of the integrated heterologous pathway.
Biosynthetic Pathway Installation
Integration of entire multi-step pathways for the synthesis of complex molecules like terpenoids, polyketides, or non-natural amino acids.
Reporter and Tag Insertion
Accurate in-frame insertion of fluorescent protein tags (e.g., GFP) or affinity tags (e.g., His-tag) onto native genes for protein localization or purification studies.
Gene Replacement/Allele Swap
Replacement of a native gene or promoter with a heterologous variant or a mutant allele (e.g., exchanging a native enzyme for an improved one).
A systematic process for construction, verification, and stable chromosomal integration.
1. Rational Design & Template Synthesis
2. Transformation and Selection
3. Marker Curing and Clone Isolation
4. Verification and Stabilization
Identify the genomic safe harbor site(s) for insertion. Design gRNA(s) for the locus.
Synthesize the gene/pathway to be inserted, ensuring codon optimization for S. cerevisiae.
Construct the large DNA donor template with optimized homology arms.
Cotransform the Cas9 system, gRNA cassette, and large DNA donor into the S. cerevisiae host.
Select for successful transformants using nutritional or drug selection markers linked to the donor template.
Isolate single colonies and expand cultures for high-throughput screening or verification.
Genotype verification via junction PCR and definitive Sanger sequencing or restriction fragment length analysis to confirm full integration.
Validate expression stability and product titer of the final engineered strain.
Delivery of the verified S. cerevisiae knock-in strain and full documentation.
Unrivaled HDR for Knock-in
Leveraging the yeast's intrinsic, high-fidelity Homology-Directed Repair system ensures the accurate integration of large, multi-gene pathways (up to 50 kb) at high efficiency.
Stable Chromosomal Integration
All integrations are placed into the chromosome, eliminating the issues of plasmid instability and copy number variation, guaranteeing reliable expression for industrial use.
Multi-Site Integration
Optimized protocols allow the simultaneous or sequential insertion of different gene copies or pathway modules into multiple safe harbor loci for maximal titer boost.
Markerless and Clean
Implementation of selection marker recycling protocols ensures the final engineered strain is free of residual resistance genes, maintaining a clean genetic background.
1. Why is yeast (S. cerevisiae) excellent for gene knock-in?
Yeast's powerful Homology-Directed Repair (HDR) pathway allows the cell to accurately incorporate large DNA fragments (the donor template) at the site of the CRISPR-Cas9 cut, resulting in very high rates of precise gene insertion.
2. What is the maximum size of DNA that can be stably integrated?
S. cerevisiae is capable of integrating pathways up to 50 kb in size in a single step using CRISPR-HDR. This makes it ideal for integrating complex biosynthetic pathways composed of multiple genes and regulatory elements.
3. What is a genomic "safe harbor" site?
A safe harbor site is a specific, non-essential region of the yeast chromosome where a heterologous gene or pathway can be integrated to ensure stable, high-level expression without interfering with the host's vital functions.
4. Can you insert multiple genes simultaneously (multiplex knock-in)?
Yes. We use multiplex gRNA systems and either assemble the entire pathway on one large donor template or use multiple donor templates to simultaneously target different genomic loci for insertion, accelerating pathway construction.
5. How does chromosomal integration benefit industrial production?
Chromosomal integration eliminates the genetic instability and variable gene dosage associated with plasmids, providing a stable copy number and reliable expression levels throughout large-scale, long-duration fermentation runs.
6. Is the insertion of the new gene permanent?
Yes. Because the gene is integrated into the stable yeast chromosome, the insertion is permanent and passed down through subsequent generations without the need for continuous selection pressure (like antibiotics).
7. What verification steps are performed for a knock-in?
We verify the knock-in using junction PCR (to confirm the new fragment is precisely integrated at the genomic boundaries) and Sanger sequencing of the integrated sequence to confirm the integrity and coding sequence of the new gene.
8. What input materials are needed to start a knock-in project?
We require the sequence of the gene or pathway to be inserted, the specific S. cerevisiae host strain, and if available, the coordinates of the preferred genomic integration site(s).
CRISPR-Cas9 technology represents a transformative advancement in gene editing techniques. The main function of the system is to precisely cut DNA sequences by combining guide RNA (gRNA) with the Cas9 protein. This technology became a mainstream genome editing tool quickly after its 2012 introduction because of its efficient, simple and low-cost nature.
The CRISPR gene editing system with its Cas9 version stands as a vital instrument for current biological research. CRISPR technology enables gene knockout (KO) through permanent gene expression blockage achieved by sequence disruption. Various scientific domains including disease modeling and drug screening employ this technology to study gene functions. CRISPR KO technology demonstrates high efficiency and precision but requires confirmation and verification post-implementation because unsatisfactory editing may produce off-target effects or incomplete gene knockouts which impact experimental result reliability. For precise and efficient Gene Editing Services - CD Biosynsis, Biosynsis offers comprehensive solutions tailored to your research needs.
The CRISPR-Cas9 knockout cell line was developed using CRISPR/Cas9 gene editing to allow scientists to remove genes accurately for research on gene function and disease models and pharmaceutical discovery. Genetic research considers this technology essential due to its high efficiency together with simple operation and broad usability.
If your question is not addressed through these resources, you can fill out the online form below and we will answer your question as soon as possible.
CD Biosynsis is a leading customer-focused biotechnology company dedicated to providing high-quality products, comprehensive service packages, and tailored solutions to support and facilitate the applications of synthetic biology in a wide range of areas.