Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Actinobacteria are crucial sources of valuable secondary metabolites.
• Genome mining suggests underestimated secondary metabolite potential in actinobacteria.
• CRISPR/Cas9 technology has revolutionized genetic manipulation in actinomycetes.

Purpose of the Study:

• To develop a more efficient CRISPR/Cas9 system for actinobacteria genome engineering.
• To streamline the screening of edited mutants and plasmid curing processes.
• To demonstrate the system's applicability in both model and non-model actinomycetes.

Main Methods:

• An updated CRISPR/Cas9 system was developed using GusA and IdgS chromogenic reporters.
• The system was tested for gene and gene cluster deletion in Streptomyces coelicolor and Verrucosispora sp.
• Efficiency was evaluated through positive clone screening and plasmid curing.

Main Results:

• The updated CRISPR/Cas9 system significantly improved the speed and efficiency of genome editing.
• Successful deletion of a single gene, a small gene cluster, and a large gene cluster was achieved.
• The system facilitated rapid screening and efficient plasmid removal.

Conclusions:

• The chromogenic reporter-based CRISPR/Cas9 system is a versatile tool for actinobacteria genome engineering.
• This approach enhances efficiency and reduces labor in genetic manipulation of actinomycetes.
• The system is broadly applicable across different actinobacterial species, including non-model organisms.