Solution for Yeast Metabolic Regulation with CRISPR/Cas9 Technology    

Saccharomyces cerevisiae (Yeast), a single-celled eukaryotic organism, is extensively studied and widely used as a major industrial microorganism in the production of bio-based chemicals. Due to its rapid growth, well-characterized genetic background, stress tolerance, ease of manipulation, and high safety, S. cerevisiae serves as an ideal model for biological research. It finds applications in various fields such as food, medicine, energy, and chemical industries. The continuous advancement of gene editing technologies and transcriptional regulation techniques has made it possible to optimize metabolic pathways and enhance the production of metabolic products.

Limitations and Developments of Traditional Gene Editing Techniques in S. cerevisiae

Traditional gene editing techniques such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have achieved some success. However, their application is limited due to the complexity of design and time-consuming processes. In contrast, the CRISPR/Cas9 system, with its high efficiency, low cost, and relative simplicity of operation, has quickly become a crucial tool in gene editing. Despite these advantages, achieving efficient, precise, and multi-site gene editing and transcriptional regulation in yeast still poses several challenges, such as low efficiency in multiple gene editing and ensuring genome stability post-editing.

CRISPR/Cas9 is an adaptive immune system capable of specifically cleaving double-stranded DNA. Initially discovered in 90% of archaea and 50% of bacteria, CRISPR/Cas9 offers several unique advantages:

1. High Efficiency: CRISPR/Cas9 can rapidly introduce gene mutations, significantly enhancing gene editing efficiency.

2. Low Cost: Compared to other gene editing technologies, the operational cost of CRISPR/Cas9 is lower, making it more suitable for large-scale applications.

3. Ease of Operation: By customizing sgRNA, the CRISPR/Cas9 system can precisely target any gene sequence of interest.

CRISPR Applications in Yeast Metabolic Regulation

In yeast, CRISPR/Cas9 has been widely used for genome editing and transcriptional regulation. The Cas9 protein, guided by sgRNA, targets specific DNA loci, introducing double-strand breaks (DSBs) to achieve gene editing. Recent advancements have led to the development of base editors and prime editors, enabling precise base substitutions and small insertions/deletions (indels) without relying on homologous recombination (HR) or non-homologous end joining (NHEJ), thereby significantly enhancing editing efficiency and accuracy.

Base Editors and Prime Editors

Base editors chemically modify bases directly, converting one base into another without inducing DSBs. For example, they can convert C to T or A to G, thus avoiding the stress response and off-target effects associated with DSBs, greatly improving editing precision and safety. Prime Editors, a more advanced tool, use pegRNA to guide reverse transcriptase to the target site, allowing for various forms of gene editing, including base substitutions, small insertions, and deletions. Prime Editors do not rely on HR or NHEJ, making them suitable for multiple gene editing applications.

Applications of CRISPR/Cas9 in Yeast Metabolic Pathway Optimization

1. Metabolic Pathway Optimization: Editing key metabolic enzyme genes can optimize metabolic pathways to increase the yield of target metabolic products. For instance, editing genes in the ethanol synthesis pathway of yeast can enhance ethanol production for industrial bioethanol production.

2. Metabolic Product Diversion: Editing branching point genes in metabolic pathways can regulate metabolic flux to direct more precursor substances toward the synthesis of target products. For example, editing genes in the fatty acid synthesis pathway can increase the yield of fatty acid derivatives for biodiesel production.

3. Metabolic Downregulation: Inhibiting the expression of negative regulatory genes can relieve inhibition of target metabolic pathways, thereby increasing the yield of target products. For instance, inhibiting negative regulatory genes in the ethanol metabolism pathway can increase ethanol accumulation.

Figure 1. Overview of (d)Cas9-based genome editing strategies in yeast, including cleavage by Cas9 guided by crRNA+tracrRNA or sgRNA, nCas9, CBEs, ABEs, Prime editor, and yEvolvR for localized random mutagenesis. (Liang Y, et al., 2024)

Solution for Yeast Metabolic Pathway Optimization

1. Design of sgRNA and Cas9 Synthesis System

• First, select suitable targets based on the gene of interest or target region. Online tools such as CRISPR Design Tool or CHOPCHOP can be used to design sgRNAs targeting specific gene sequences, predicting off-target effects, and selecting optimal sgRNA sequences.
• Once the sgRNA sequence is determined, it can be obtained through chemical synthesis or PCR amplification and then cloned into an appropriate expression vector, usually a high-copy plasmid, to ensure adequate expression levels.
• Select the appropriate Cas9 variant based on specific experimental needs. For example, conventional SpCas9 is suitable for most gene editing needs, while enhanced versions such as eSpCas9 or HypaCas9 offer higher specificity and reduced off-target effects.
• To improve editing efficiency, introduce a negative selection marker gene (e.g., CAN1). A negative selection of cells that are not successfully edited ensures that only successfully edited cell strains are retained.

2. Multi-site Gene Editing

• Include multiple sgRNA expression cassettes in a single plasmid so that multiple sgRNAs can be simultaneously expressed, targeting different gene loci. This can be achieved by using various promoters (e.g., U6 and tRNA promoters) to drive the expression of each sgRNA.
• Integrate multiple sgRNAs and Cas9 genes into a single plasmid to ensure coordinated expression. This integration plasmid can be constructed through methods such as Golden Gate assembly or Gibson assembly.
• By adding self-cleaving sequences (e.g., Csy4 recognition sites) between sgRNAs, multiple sgRNAs can be efficiently co-expressed. Additionally, multiple sgRNA sequences can be spliced together within a single expression cassette, forming a multi-sgRNA tandem post-transcription, which can then be processed by RNA processing enzymes to generate individual sgRNAs.
• Adding RNA aptamer structures to the ends of sgRNAs enhances their stability and functional activity within cells, thus improving editing efficiency.

3. Gene Transcription Regulation

• Using deactivated Cas9 protein (dCas9), which binds to the target gene promoter region without causing DNA breaks. dCas9 can be fused with transcriptional repressors (e.g., KRAB) or activators (e.g., VP64) to achieve gene downregulation or upregulation.
• Combining RNA aptamers and optogenetics enables precise spatial and temporal control. For instance, optogenetic tools can activate or inhibit dCas9 activity through light exposure, achieving temporally precise gene expression regulation.

4. Optimization of System Expression and Localization

• Select strong and suitable promoters for yeast (e.g., TEF1, PGK1) to drive Cas9 and sgRNA expression, ensuring adequate expression levels.
• Adding nuclear localization signals (NLS) to Cas9 and dCas9 proteins ensures efficient nuclear entry, enhancing editing and regulation efficiency.
• Optimizing Cas9 expression levels and selecting appropriate expression vectors can reduce cytotoxicity caused by Cas9 overexpression. An inducible expression system (e.g., GAL1 promoter) can be used to control Cas9 expression.

5. Application of Prime Editor (PE) System

• Prime Editor consists of a Cas9 variant fused with reverse transcriptase and pegRNA. PegRNA contains not only the sgRNA sequence but also an editing template sequence, guiding reverse transcriptase to insert the editing sequence at the target site.
• The PE system can achieve high-precision base substitutions and small insertions/deletions (indels) without introducing DSBs. This reduces nonspecific mutations caused by NHEJ, enhancing editing specificity and precision.
• Unlike traditional homologous recombination repair (HR), the PE system does not require exogenous donor DNA, greatly simplifying the editing process and reducing the risk of random donor DNA integration.

Creative Biogene: Leading the Way in CRISPR/Cas9 Solutions

Looking ahead, with ongoing advancements and innovations in CRISPR/Cas9 technology, the potential for yeast metabolic regulation is immense. The continuous refinement of these techniques promises to overcome existing challenges and push the boundaries of efficiency, precision, and control in both industrial and research applications.

Creative Biogene specializes in harnessing the power of the CRISPR/Cas9 system to drive innovation in yeast applications. Our advanced CRISPR/Cas9 solutions are designed to support your research and industrial needs, providing robust tools for metabolic engineering, pharmaceutical research, and fundamental biological studies. Partner with Creative Biogene and explore new frontiers in biotechnology with precision and efficiency.