Ribonucleoprotein-based CRISPR/Cas9 genome co-editing in Aspergillus luchuensis mut. kawachii

Area of Science:

• Microbiology
• Molecular Biology
• Genetic Engineering

Background:

• Aspergillus luchuensis mut. kawachii is a fungus with industrial importance.
• Efficient genome editing tools are crucial for genetic manipulation of A. luchuensis mut. kawachii.

Purpose of the Study:

• To establish a ribonucleoprotein-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome co-editing method for A. luchuensis mut. kawachii.
• To optimize protoplast preparation for efficient delivery of CRISPR/Cas9 components.
• To demonstrate the applicability of the method for gene knockouts and co-editing in both model and industrial strains.

Main Methods:

• Optimization of protoplast preparation using Yatalase -Plus-.
• Introduction of single guide RNA-Cas9 ribonucleoprotein complexes into A. luchuensis mut. kawachii protoplasts.
• Gene knockout of the ATP sulfurylase-encoding sC gene.
• Genome co-editing targeting sC with pyrG or prtR genes.

Main Results:

• Successful optimization of protoplast preparation for A. luchuensis mut. kawachii.
• Efficient knockout of the sC gene in both NBRC 4308 and No. 8046 strains.
• Successful genome co-editing to generate pyrG and prtR knockout strains in NBRC 4308.
• Successful generation of a prtR knockout strain in the industrial No. 8046 strain.

Conclusions:

• The ribonucleoprotein-based CRISPR/Cas9 genome co-editing method is effective for A. luchuensis mut. kawachii.
• This method is applicable to both model and industrial strains, offering a versatile tool for genetic manipulation.
• The developed method holds promise for future research and applications in A. luchuensis mut. kawachii.