A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Molecular genetics

Background:

• Pichia pastoris is a key microbial cell factory for producing proteins and chemicals.
• Limited genome engineering tools hinder Pichia pastoris applications in metabolic engineering and synthetic biology.

Purpose of the Study:

• To develop a versatile CRISPR-based toolbox for gene editing and transcriptional regulation in Pichia pastoris.
• To enhance Pichia pastoris capabilities for advanced metabolic engineering and synthetic biology.

Main Methods:

• Constructed a CRISPR/Cas9 system for gene editing with RNA Pol-III-driven sgRNA expression and an eliminable episomal plasmid.
• Developed CRISPR/dCas9 tools fused with transcriptional repressors (Mix1/RD1152) or activators (VPR) for gene expression regulation.
• Engineered a CRISPR-ARE system for simultaneous gene activation, repression, and editing using multiplex sgRNAs.

Main Results:

• Achieved up to 100% gene knockout efficiency using the CRISPR/Cas9 system.
• Demonstrated strong gene repression (above 70%) and activation (up to 3.5-fold) of reporter genes.
• Showcased the CRISPR-ARE system's efficiency in individual and combinatorial gene activation, repression, and editing.

Conclusions:

• The developed CRISPR toolbox provides simple and multifunctional tools for Pichia pastoris.
• This toolkit will significantly accelerate Pichia pastoris applications in metabolic engineering and synthetic biology.
• The system enables precise genetic manipulation for optimizing microbial cell factories.