Mie-Streuung
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.