An improved CRISPRi system in Pichia pastoris

Area of Science:

• Molecular Biology
• Synthetic Biology
• Microbial Biotechnology

Background:

• CRISPR interference (CRISPRi) is a powerful tool for gene repression in diverse organisms.
• Optimizing CRISPRi systems in yeast, such as Pichia pastoris, is crucial for metabolic engineering and functional genomics.

Purpose of the Study:

• To develop and enhance a CRISPRi system in Pichia pastoris for robust gene repression.
• To investigate the impact of guide RNA (gRNA) processing on CRISPRi efficiency.
• To demonstrate multiplexed gene repression using an engineered CRISPRi system.

Main Methods:

• Fusion of dCas9 with endogenous transcriptional repressor domains in Pichia pastoris.
• Targeting the AOX1 promoter and eGFP gene for repression.
• Utilizing HH/HDV ribozymes and Csy4 endoribonuclease for gRNA processing.
• Constructing gRNA arrays for simultaneous repression of multiple genes.

Main Results:

• Achieved up to 85% repression efficiency for eGFP.
• Demonstrated efficient repression of the native AOX1 gene, impacting growth on methanol.
• Showed that Csy4-mediated gRNA processing yields higher repression efficiency compared to native tRNA systems.
• Successfully repressed multiple genes (e.g., FAA1 and FAA2) simultaneously using Csy4-processed gRNA arrays.

Conclusions:

• The enhanced CRISPRi system in Pichia pastoris provides strong and tunable gene repression.
• Csy4 endoribonuclease is effective for processing gRNA arrays, enabling efficient multiplexed gene repression.
• This improved system offers a valuable tool for genetic manipulation and synthetic biology in Pichia pastoris.