Custom Yeast Gene Editing

Yeast Multi-Gene Knockout Strain Construction

CRISPR/Cas9-mediated sequential or simultaneous knockout of multiple genes in Saccharomyces cerevisiae for functional genomics and metabolic engineering.

Custom gRNA design for 2–10+ target genes per project
Marker recycling workflow enables sequential editing
PCR and Sanger sequencing confirmation of each deletion

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Service Highlights

Efficient CRISPR/Cas9 system tailored for multi-gene knockout in S. cerevisiae, with scalable workflow and full sequence verification.

Iterative editing with marker recycling up to 10+ rounds
In silico off-target prediction and optional targeted confirmation
Delivered as validated strain(s) with quality documentation

CRISPR/Cas9

S. cerevisiae

Multi-Gene Knockout

Our Approach

We combine custom guide RNA design, high-efficiency transformation, and a marker-recycling strategy to disrupt multiple genomic loci in Saccharomyces cerevisiae sequentially or in parallel. This workflow is tailored for projects requiring 2–10+ gene knockouts in a single strain, enabling systematic functional analysis and metabolic reprogramming.

Guide RNA Design

Custom gRNAs designed against user-specified target sequences, including multiple isoform considerations.

Transformation & Selection

High-efficiency transformation of S. cerevisiae with CRISPR plasmids; colonies screened on selective media.

Screening & Validation

Colony PCR spanning each deletion junction followed by Sanger sequencing to confirm complete knockout.

Deliverables

What You Receive

All deliverables are scoped at project start and provided with project documentation.

Standard Deliverables

Validated multi-gene knockout strain(s) (glycerol stock and streak)
Sanger sequencing data for each deleted locus
GRNA expression plasmids (as scoped)
Project report: design, methods, and QC results

Additional Options (when scoped)

PCR gel images and raw sequencing trace files
Marker recycling documentation
Off-target analysis report (in silico + targeted amplicon sequencing)
Glycerol stocks of intermediate strains

Service Scope

Project Parameters

Our service is tailored to your specific targets. The table below outlines typical scoping parameters; all details are finalized during consultation.

Parameter	Typical project scope
Host Strain	Saccharomyces cerevisiae (lab strains; customer-provided strain considered case-by-case)
Number of Target Genes	2 to 10+ genes per project; sequential or simultaneous editing strategy defined at design
gRNA Design	Custom design using validated algorithms; on-target scoring and off-target prediction provided
Transformation Method	Lithium acetate/PEG high-efficiency protocol; alternative methods considered on request
Selectable Markers	Auxotrophic (e.g., URA3, LEU2) or antibiotic (e.g., G418, hygromycin) as scoped; marker recycling available for multi-round editing
Validation	Colony PCR for each deletion junction; Sanger sequencing of PCR amplicons. Off-target analysis by in silico prediction + optional targeted amplicon sequencing

Workflow

How It Works

From target selection to validated strain — a structured 5-step process.

Target Selection & gRNA Design

You provide gene names or sequences; our bioinformatics team designs optimized guide RNAs for each target, including off-target scoring. Designs are reviewed with you before proceeding.

CRISPR Plasmid Construction

Each gRNA is cloned into a Cas9 expression vector. Plasmids are sequence-verified and prepared in sufficient quantity for transformation.

Yeast Transformation & Selection

Competent yeast cells are transformed with the CRISPR plasmid using a lithium acetate/PEG protocol. Transformed cells are plated on selective medium and incubated until colonies appear.

Marker Recycling (if sequential)

For multi-round editing, the marker is excised from the genome (e.g., by flanking loxP sites or homologous recombination), enabling its reuse in the next deletion round.

Clone Isolation & Verification

Individual colonies are picked, genomic DNA is extracted, and each target locus is amplified by PCR. Sanger sequencing confirms the intended knockout; verified clones are cryopreserved.

Applications

Functional Genomics

Investigate gene function and genetic interactions by systematically deleting multiple genes. Identify synthetic lethal pairs or pathway redundancy.

Gene function and essentiality studies
Genetic interaction mapping
Pathway and network analysis

Metabolic Engineering

Disrupt specific metabolic routes or regulatory elements to study impacts on carbon flux and product formation in yeast.

Metabolic pathway evaluation
By-product reduction studies
Target metabolite production assessment

Synthetic Biology

Construct minimal genomes or engineer strains with designer modules by removing non-essential genes while retaining robust growth.

Genome streamlining and minimal cell design
Chassis strain development
Heterologous pathway integration

FAQs

Frequently Asked Questions

Common questions about our multi-gene knockout service.

Project duration depends on the number of targets, sequence complexity, and whether screening is done sequentially or in parallel. A typical 3–5 gene knockout project may require a timeline confirmed during project scoping, including design, transformation rounds, and validation. An exact timeline is determined during consultation and depends on target biology and available resources.
We routinely perform knockouts of 2–10 genes. For larger sets, we recommend a sequential strategy with marker recycling. Projects with more than 10 targets can be scoped on a case-by-case basis, and we work with you to investigate most efficient editing order and selection scheme.
Each deletion is verified by colony PCR across the target locus and Sanger sequencing of the PCR product. We also provide in silico off-target prediction for each gRNA. Optional targeted amplicon sequencing for predicted off-target sites can be included in the scope. All QC data is supplied with the final strain.
Yes, we can accept customer-provided S. cerevisiae strains, provided they are amenable to our transformation protocol (competent cell preparation). Strain background and any pre-existing markers are evaluated during the design phase to support compatibility with the multi-gene editing workflow.

Scientific Literature

References

Feng Y-H, Song J-Z, Zhu J, et al. Template plasmids optimized for deletion of multiple genes in yeast Saccharomyces cerevisiae. Microbiology spectrum. 2024;12(11):e0132024. View on PubMed
Tian T, Yang C, Wu JL, et al. Zn(2+) supplementation-mediated IZH4 enhances acid tolerance in Saccharomyces cerevisiae via membrane lipid remodeling: A multi-omics study. Food chemistry. Molecular sciences. 2025;11:100324. View on PubMed
Gong X, Shen Y, Shang S, et al. An unfolded protein response-inducible protein (ULI1) mediates aromatic alcohols biosynthesis through reinforcing the Ehrlich pathway in Saccharomyces cerevisiae. Frontiers in microbiology. 2026;17:1934502. View on PubMed

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