One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 technology facilitates targeted genome modifications in various bacteria.
• Efficient genetic manipulation tools are crucial for understanding and engineering Streptomyces species.

Purpose of the Study:

• To develop a high-efficiency CRISPR/Cas9 genome editing system for Streptomyces.
• To demonstrate its utility in deleting single genes, large gene clusters, and introducing point mutations.

Main Methods:

• Construction of the pKCcas9dO plasmid containing guide RNA, codon-optimized Cas9, and homology-directed repair templates.
• Delivery of editing plasmids into Streptomyces coelicolor M145 via intergeneric transfer.
• Application of the system for gene deletion, gene cluster deletion, and point mutation introduction.

Main Results:

• Achieved high genome editing efficiencies (60%-100%) for single gene and large gene cluster deletions.
• Successfully performed simultaneous deletions of multiple genes and gene clusters with efficiencies of 54% and 45%.
• Introduced streptomycin resistance mutations by altering the rpsL gene.

Conclusions:

• The developed CRISPR/Cas9 system significantly enhances genome editing efficiency in Streptomyces.
• This method reduces the time required for genome modification by one-third to one-half.
• The system shows promise for genome engineering in other Actinomyces species.