Yeast CRISPRi Gene Repression Service

The Yeast CRISPRi (CRISPR Interference) Gene Repression Service offers a powerful, rapid, and reversible method for downregulating (silencing) the expression of endogenous genes in yeast strains like Saccharomyces cerevisiae. Unlike traditional gene knockout, CRISPRi uses a catalytically dead Cas9 (dCas9) fused to a repressor domain, which is guided to a target gene's promoter region by a guide RNA (gRNA), blocking transcription.

CD Biosynsis provides a specialized CRISPRi platform that allows for tunable repression—the degree of gene silencing can be precisely controlled by adjusting the inducer concentration. This service is invaluable for metabolic engineering to balance pathway flux, identify rate-limiting steps without lethal knockouts, and study the effects of gene dosage on bioprocess performance. We deliver chromosomally integrated, stable CRISPRi strains with verified repression capacity, offering superior control compared to transient methods.

Highlights

Key advantages of using CRISPRi for gene silencing in yeast:

• Tunable Repression: Allows for graded gene silencing (partial to near-complete knockdown) by controlling the inducer concentration, which is critical for flux balancing.
• Reversible Control: Repression can be turned off (or on) rapidly, enabling time-course studies of essential genes or dynamic control during fermentation.
• Non-Lethal for Essential Genes: Provides a method to study the function of essential genes where a complete knockout would be lethal to the cell.
• Multiplex Interference: Multiple genes can be repressed simultaneously using a set of orthogonal gRNAs, facilitating complex pathway analysis.

Repression Applications

Applications benefiting from precise and tunable gene repression in yeast:

Metabolic Flux Balancing

Fine-tuning the expression of native metabolic enzymes to optimize the flow of carbon towards a desired product.

Essential Gene Studies

Studying the phenotypic consequences of partially silencing genes critical for cell viability and growth.

Bypass Toxicity

Transiently repressing genes related to growth or stress to allocate cellular resources toward high-value product synthesis.

Target Validation and Screening

Quickly assessing the impact of silencing multiple putative target genes before committing to permanent knockout strategies.

Key Features & Tunability

Technical features of our optimized Yeast CRISPRi platform:

Inducible dCas9 System

Chromosomally integrated dCas9 fused to a repressor, controlled by an inducible promoter (e.g., Tet-on or galactose), offering tunable control.

Optimized gRNA Design

Proprietary guide RNA design rules targeting the Transcription Start Site (TSS) for maximal repression efficiency.

Multiplex Repression

Ability to co-express multiple gRNAs to simultaneously repress up to five different genes for complex metabolic network tuning.

Host Compatibility

CRISPRi platform is adaptable to S. cerevisiae, P. pastoris, and other non-conventional yeast hosts.

Stability and Curing

The core dCas9 system is chromosomally integrated for stability, while the gRNA plasmid can be transiently used or also integrated.

Workflow

Our systematic workflow for constructing stable Yeast CRISPRi strains:

• dCas9 Integration: Chromosomal integration of the dCas9-repressor fusion protein under a tightly controlled inducible promoter into the target yeast strain.
• gRNA Design and Cloning: Design high-efficiency gRNAs targeting the promoter or coding sequence region of the target gene(s).
• gRNA Delivery: Introduce the gRNA expression cassette (plasmid or integrated) into the dCas9-containing host strain.
• Verification of Repression: Perform controlled induction experiments followed by qPCR analysis to quantify the level of mRNA repression.
• Phenotypic Analysis (Optional): Test the engineered strain under various induction levels to map the relationship between repression strength and phenotype (e.g., titer).

We provide essential assurance for high-quality CRISPRi repression outcomes:

• Guaranteed Repression: Commitment to deliver a verified strain exhibiting a defined level of target mRNA repression (e.g., >70 percent knockdown).
• Tunable Control: Provision of data demonstrating the ability to modulate gene expression level by varying the inducer concentration.
• Stable System: Assurance that the core dCas9 repression machinery is stably integrated into the chromosome for reliable long-term performance.
• Target Specificity: Rigorous gRNA design to minimize off-target effects and ensure specific gene silencing.