Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia...

Area of Science:

• Biotechnology
• Synthetic Biology
• Molecular Biology

Background:

• Yarrowia lipolytica is a key yeast for high-level lipid accumulation.
• Existing genetic tools for Y. lipolytica require improvement for rapid strain generation.

Purpose of the Study:

• To develop a versatile CRISPR-Cpf1 system for simultaneous gene disruption and regulation in Y. lipolytica.
• To enable rapid strain engineering for biotechnological applications.

Main Methods:

• Engineered a CRISPR-Cpf1 system with truncated guide RNAs (16 nt) for gene regulation.
• Utilized Cpf1-Mxi1 fusion for gene repression and Cpf1-VPR fusion for CRISPR-activation.
• Employed longer guide RNAs (23-25 bp) for efficient gene disruption.
• Demonstrated multiplexed expression of truncated and full-length guide RNAs.

Main Results:

• Achieved 7-fold mRNA reduction via Cpf1-Mxi1-mediated gene repression.
• Increased hrGFP expression 10-fold using Cpf1-VPR-mediated CRISPR-activation.
• Demonstrated high-efficiency gene disruptions with specific guide RNA lengths.
• Maintained disruption and repression efficiencies with multiplexed guide RNA expression.

Conclusions:

• The developed CRISPR-Cpf1 system offers dual functionality for gene editing and regulation.
• This system facilitates rapid strain generation in Y. lipolytica.
• The tool is valuable for metabolic engineering, genome-wide screening, and functional genomics.