Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1.

Area of Science:

• Microbial biotechnology
• Molecular biology
• Synthetic biology

Background:

• CRISPR/Cas9 is crucial for fungal genome engineering in Ashbya gossypii.
• The Cas9 system's utility is limited by its specific 5'-NGG-3' PAM sequence requirement.
• A broader range of genome editing tools is needed for Ashbya gossypii.

Purpose of the Study:

• To introduce a novel CRISPR/Cpf1 system for Ashbya gossypii genome engineering.
• To overcome the PAM limitations of the CRISPR/Cas9 system.
• To develop a multiplex system for efficient gene deletion in Ashbya gossypii.

Main Methods:

• Adaptation of the CRISPR/Cpf1 system using Lachnospiraceae bacterium's Cpf1 nuclease.
• Utilizing a T-rich PAM sequence (5'-TTTN-3') for target site recognition.
• Implementation of a multiplex CRISPR/Cpf1 system with crRNA and dDNA arrays for gene deletion.

Main Results:

• The CRISPR/Cpf1 system successfully introduced large deletions in Ashbya gossypii.
• Validation was performed using five auxotrophic markers (HIS3, ADE2, TRP1, LEU2, URA3).
• Multiplex gene deletion of up to four genes was achieved using a single multi-CRISPR/Cpf1 plasmid.

Conclusions:

• The CRISPR/Cpf1 system significantly expands the genome engineering toolbox for Ashbya gossypii.
• This system enables efficient multiplex gene editing, facilitating advancements in fungal biotechnology.
• Target sequence selection is a critical factor influencing the editing efficiency of CRISPR systems.