A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts

Area of Science:

• Molecular Biology
• Microbiology
• Biotechnology

Background:

• Methylotrophic yeasts, including Ogataea polymorpha and Komagataella phaffii, are vital for research and industry, often needing precise genome modifications.
• Existing CRISPR-Cas9 methods for these yeasts are complex and time-consuming, hindering their widespread application.

Purpose of the Study:

• To develop an improved and simplified plasmid vector set for efficient CRISPR-Cas9 genome editing in methylotrophic yeasts.
• To facilitate easier and faster genome modifications in O. polymorpha and K. phaffii.

Main Methods:

• Designed a CRISPR-Cas9 vector set featuring a Cas9-encoding plasmid with a nuclear localization signal and sgRNA scaffold plasmids.
• Implemented a novel recombination-based selection system for autonomous plasmid formation and subsequent loss.
• Utilized G418-resistance and LEU2 auxotrophic markers for selection.

Main Results:

• Successfully demonstrated the functionality of the improved CRISPR-Cas9 system in multiple methylotrophic yeast species: O. polymorpha, O. parapolymorpha, O. haglerorum, and K. phaffii.
• The vector set simplifies sgRNA gene construction via oligonucleotide insertion.
• The recombination-based system ensures plasmid loss after genome editing is complete.

Conclusions:

• The developed plasmid vector set significantly improves the ease and efficiency of CRISPR-Cas9 genome editing in methylotrophic yeasts.
• This advancement provides a valuable tool for both basic research and biotechnological applications involving these industrially relevant yeasts.