CRISPR/Cas9 Genome Editing

Saccharomyces cerevisiae Multi-Gene Knockout Strain Construction Service

Rapidly generate validated multi-gene knockout strains in S. cerevisiae using a multiplex CRISPR/Cas9 system.

Simultaneous knockout of multiple target genes in Saccharomyces.
Custom gRNA design and selection marker options.
Validated strains delivered with full genotyping documentation

Streamlined multiplex gene editing for yeast

Our service combines CRISPR/Cas9‑mediated genome editing with marker recycling to enable efficient, iterative disruption of multiple genes in Saccharomyces cerevisiae. Ideal for functional genomics, metabolic engineering, and synthetic biology applications.

Multiplex gRNA design and construction
Auxotrophic or drug‑selection markers
PCR and Sanger sequencing verification per target

Custom gRNA design

Marker recycling included

Monoclonal strain isolation

How It Works

Multiplex CRISPR/Cas9 knockout in S. cerevisiae

A stepwise process combining custom gRNA design, plasmid‑based Cas9 delivery, and iterative marker recycling to achieve clean multi‑gene deletions.

GRNA Design and Plasmid Construction

We design multiple guide RNAs targeting your genes of interest using proprietary algorithms that minimize off‑target potential. Each gRNA is cloned into a CRISPR/Cas9 expression plasmid along with a selection marker (auxotrophic or drug resistance).

Up to 6 gRNAs per round (scope‑dependent)
Choice of marker: URA3, LEU2, HIS3, or drug cassettes
In silico off‑target assessment

Transformation, Selection, and Marker Recycling

Yeast strains are transformed with the CRISPR plasmid(s) and selected on appropriate media. For sequential knockouts, the marker is recycled using counter‑selection or Cre‑lox recombination, allowing reuse of the same marker for the next round.

Lithium acetate or electroporation transformation
Colony PCR to confirm marker insertion
Marker excision verified by PCR

Customizable Scope

Number of targets, marker selection, and editing strategy are scoped per project. We accommodate both single‑round multiplex and iterative approaches.

Rigorous Validation

Each deletion is confirmed by colony PCR and Sanger sequencing. The final strain is purified to monoclonal status and archived.

Deliverables Ready for Downstream Use

Receive validated strain(s), plasmid constructs, and a detailed QC report — all structured to integrate into your research or production pipeline.

What You Receive

Validated Multi‑Gene Knockout Strain Package

Each project delivers a fully documented, monoclonal knockout strain suitable for immediate use.

Strain & Reagents

One or more purified Saccharomyces cerevisiae knockout strains (quantity as scoped)
CRISPR/Cas9 expression plasmids and gRNA constructs used
Selection marker cassettes (if applicable)

Documentation

Genotyping report with PCR gel images and Sanger sequencing chromatograms for each target.
In silico off‑target analysis summary
Strain stability and purity certificate (monoclonal origin confirmed)

Project Parameters

Scoped Capabilities

All parameters are defined during project design; the table below outlines typical ranges.

Parameter	Typical project scope
Number of target genes	As scoped; typically 2–6 per project, scalable via iterative rounds
Editing strategy	Gene disruption via indel or complete open reading frame deletion
Selection markers available	Auxotrophic (URA3, LEU2, HIS3, TRP1) or drug resistance (G418, nourseothricin)
Marker recycling	Included for sequential knockouts (Cre‑lox or 5‑FOA counter‑selection)
Verification method	Colony PCR + Sanger sequencing for each edited locus
Off‑target analysis	In silico prediction; targeted amplicon sequencing when scoped
Strain purification	Single‑colony isolation to support monoclonal genotype
Technical support	Named scientific contact at project start; milestone review calls; email response within 1 business day

Engagement Process

From Gene Targets to Validated Strain

A streamlined, five‑stage workflow that keeps you informed at every step.

Gene Target Selection & gRNA Design

You provide the list of target genes (or we help identify candidates). We design gene‑specific guide RNAs using algorithms that balance on‑target efficiency and minimize potential off‑target activity, then confirm each gRNA target sequence in your strain background.

Plasmid Construction & Transformation

Each gRNA is cloned into a CRISPR/Cas9 expression plasmid carrying a selection marker. The plasmid(s) are transformed into Saccharomyces cerevisiae using an optimized protocol (lithium acetate or electroporation) and plated on selective media.

Selection & Colony Screening

Transformant colonies are screened by colony PCR to identify candidates with the desired edit. Positive clones are streaked for purification and subjected to additional PCR verification across each target locus.

Marker Recycling (Iterative Editing)

When multiple rounds of editing are needed, the selection marker is excised (e.g., Cre‑lox or 5‑FOA counter‑selection). The marker‑free strain is then used for the next round of transformation with new gRNA plasmids.

Final Validation & Delivery

The final monoclonal strain is confirmed by PCR and Sanger sequencing for all edited loci. A comprehensive QC report including gel images, chromatograms, and off‑target summary is prepared. The validated strain and associated plasmids are shipped.

Research Applications

Where Multi‑Gene Knockout Strains Are Used

Our service supports a broad range of basic and applied yeast research.

Gene Function & Genetic Interactions

Systematically disrupt multiple genes to study synthetic lethality, genetic interaction networks, and pathway redundancy in Saccharomyces cerevisiae.

Essentiality and buffering studies
Epistasis mapping
Signaling pathway dissection

Metabolic Pathway Rewiring

Eliminate competing pathways or regulatory repressors to redirect carbon flux toward desired products such as ethanol, lactate, or advanced biofuels.

Deletion of by‑product genes
Elimination of negative regulators
Multi‑gene perturbation of central metabolism

Genome Minimization & Chassis Construction

Create minimal genome strains by knocking out non‑essential genes, providing a cleaner host for synthetic circuits or heterologous pathway integration.

Stepwise deletion of non‑essential regions
Reduced genomic complexity
Defined chassis for BioBrick assembly

Common Questions

Frequently Asked Questions

Practical answers about our multi‑gene knockout service.

The number of targets is scoped per project and depends on factors such as gene essentiality, editing strategy, and desired timeline. Typical projects involve 2–6 genes, with iterative rounds enabling larger sets. We will discuss feasibility during the initial consultation.
Every edited locus is confirmed by colony PCR and Sanger sequencing.The final strain is purified to monoclonal origin and re‑screened to support genotype stability.
Yes. We routinely handle S. cerevisiae laboratory strains (e.g., BY4741, W303, CEN.PK, S288C derivatives). For other backgrounds, a preliminary transformation efficiency test may be performed. Please provide strain details during project scoping.
Project duration depends on the number of target genes and the chosen editing strategy. A project involving 2–3 genes with one round of marker recycling generally completes a timeline confirmed during project scoping. Exact timelines are provided in the project proposal.

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
Zhang ZX, Wang LR, Xu YS, et al. Recent advances in the application of multiplex genome editing in Saccharomyces cerevisiae. Applied microbiology and biotechnology. 2021;105(10):3873-3882. View on PubMed

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