Yeast Genome Engineering

CRISPR/Cas9 Yeast Gene Knockout Service

Permanent gene disruption in Saccharomyces cerevisiae via targeted CRISPR/Cas9 editing for functional genomics and strain engineering.

Targeted gene knockout design
Validated isogenic knockout strains
Comprehensive QC report with sequencing confirmation

Our service provides custom CRISPR/Cas9-mediated knockout of non-essential genes in Saccharomyces cerevisiae using guide RNA design and plasmid-based editing. We employ selection markers such as auxotrophic or drug resistance to isolate edited clones, and offer both single and double knockout options depending on project requirements.

Guide RNA design and synthesis
Cas9 plasmid and transformation
Selection and clone isolation

Gene Knockout

S. cerevisiae

CRISPR/Cas9

Our Approach

Our approach

Custom Guide Design

Guide RNA sequences are designed against the target gene, with in silico off-target evaluation and sequence verification of the assembled constructs.

Individual sgRNA design
Off-target evaluation
Sequence-verified constructs

Transformation & Screening

Delivery of CRISPR components into S. cerevisiae followed by selection and colony screening.

High-efficiency transformation
Selection marker assessment
PCR-based genotype screening

Deliverables

What You Receive

Each project delivers documented knockout strains and associated data.

Strains & Samples

Isogenic knockout clone(s) in glycerol stock
Wild-type control strain
Cryopreserved stock (2 vials per clone)

Data & Reports

Design report with guide RNA sequences
Screening results (PCR, gel images)
Sequencing confirmation of target locus
QC summary including editing outcome

Service Scope

Parameter	Typical project scope
Host strain	Saccharomyces cerevisiae (e.g., BY4741, BY4742, or customer-provided)
Gene target	Single non-essential open reading frame; essential genes require conditional approach
Editing modality	CRISPR/Cas9 knockout (NHEJ or HDR deletion)
Guide RNAs designed	2-3 per target (as scoped)
Selection marker	Auxotrophic (e.g., URA3, HIS3) or drug resistance (e.g., G418) as per design
Knockout type	Single or double gene knockout, scoped per project
Screening depth	Scoped per project after consultation.
Validation method	PCR junction analysis + Sanger sequencing
Turnaround time	Scoped per project after consultation
Technical support	Named scientific contact at project start; milestone review calls; email response within 1 business day

Workflow

How It Works

A streamlined process from gene target to validated knockout strain.

Gene Target Analysis

Review your gene of interest and design guide RNA sequences using in silico tools. Evaluate off-target potential and select optimal guides for transformation.

CRISPR Construct Assembly

Clone guide RNA into Cas9 expression plasmid and prepare transformation-competent yeast cells. Include selection marker for positive clone identification.

Yeast Transformation & Selection

Deliver CRISPR plasmid into S. cerevisiae via lithium acetate transformation. Plate on selective media and incubate for colony formation.

Clone Screening & Genotyping

Pick individual colonies, extract genomic DNA, and perform PCR across the target locus. Identify clones with anticipated deletion or disruption.

Validation & Cryopreservation

Confirm editing by Sanger sequencing of the target region. Purify validated knockout clones, prepare glycerol stocks, and generate final QC report.

Applications

Gene Function Discovery

Knockout strains are foundational for linking genotype to phenotype in yeast.

Phenotypic screening
Epistasis analysis
Essentiality studies

Pathway Modulation

Gene disruption to investigate metabolic pathway roles.

Target gene deletion
Selection marker assessment
Sequencing confirmation

Chassis Engineering

Create stable auxotrophic or marker-less backgrounds for synthetic constructs.

Auxotrophic marker removal
Genome reduction
Standardized background strains

FAQ

Frequently Asked Questions

Common inquiries about our yeast knockout service.

Essential genes require conditional systems such as inducible promoters or diploid knockouts with heterozygous deletion. We can discuss options for essential gene targeting on a per-project basis.
The timeline from target sequence approval to project completion is confirmed during project scoping. It depends on transformation efficiency and screening complexity.
Yes, we accept customer-provided strains. We confirm transformation compatibility and adjust selection markers as needed to support successful editing.
We use PCR to amplify the target region and Sanger sequencing to confirm the intended deletion or disruption. For quality control, we also include wild-type controls.

Scientific Literature

References

Li J, Wu T, Wang J, et al. Dual auxotrophy coupled red labeling strategy for efficient genome editing in Saccharomyces cerevisiae. Fungal genetics and biology: FG & B. 2024;173:103910. View on PubMed
Al-Fahad AJ, Al-Fageeh MB, Kharbatia NM, et al. Metabolically engineered recombinant Saccharomyces cerevisiae for the production of 2-Deoxy-scyllo-inosose (2-DOI). Metabolic engineering communications. 2020;11:e00134. View on PubMed
Huang T, Jiang G, Zhang Y, et al. The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy. EMBO reports. 2022;23(11):e54993. View on PubMed
Ramakrishnan P, Keeney J. The yeast gene ECM9 regulates cell wall maintenance and cell division in stress conditions. microPublication biology. 2024;2024. View on PubMed
Tonsager AJ, Stargell LA. An undergraduate research experience in CRISPR-Cas9 mediated eukaryotic genome editing to teach fundamental biochemistry techniques. Biochemistry and molecular biology education: a bimonthly publication of the International Union of Biochemistry and Molecular Biology. 2025;53(1):33-45. View on PubMed

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