Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a

Area of Science:

• Molecular Biology
• Synthetic Biology
• Gene Regulation

Background:

• CRISPR Cas12a is an RNA-programmable endonuclease with potential for gene regulation.
• Its T-rich PAM preference aids targeting AT-rich promoters.
• Intrinsic RNase activity simplifies multiplexed gene regulation by processing CRISPR RNA arrays.

Purpose of the Study:

• To develop and evaluate a flexible deactivated Cas12a (dCas12a)-based CRISPR interference (CRISPRi) system in Saccharomyces cerevisiae.
• To systematically assess key design features for optimal CRISPRi system performance.
• To enable precise and efficient gene regulation in yeast.

Main Methods:

• Development of a dCas12a-based CRISPRi system in S. cerevisiae.
• Systematic evaluation of NLS position, repression domains (Mxi1, MIG1), and gRNA target site.
• Optimization of dCas12a E925A variant with a C-terminal NLS.
• Implementation of inducible regulation using an RNAP II promoter.
• Demonstration of multiplexed gene regulation for a heterologous pathway.

Main Results:

• An optimized dCas12a CRISPRi system achieved up to 97% reporter gene downregulation.
• The system incorporates a single C-terminal NLS and Mxi1 or MIG1 repression domains.
• Inducible and multiplexed gene regulation strategies were successfully implemented.
• Position-dependent effects in crRNA arrays were observed.

Conclusions:

• The study provides valuable insights into dCas12a-based CRISPRi design constraints in S. cerevisiae.
• The developed system offers a flexible and efficient tool for gene regulation.
• This work enables new possibilities for precise genetic engineering in yeast.