CRISPR-Cas9 Based Engineering of Actinomycetal Genomes

Area of Science:

• Microbiology and Molecular Biology
• Biotechnology and Genetic Engineering

Background:

• Actinomycetales bacteria are crucial sources of pharmacologically active and industrially relevant secondary metabolites.
• Genetic manipulation of many actinomycetes is challenging, hindering metabolic engineering efforts.
• Efficient tools are needed to overcome these limitations and unlock the biosynthetic potential of actinomycetes.

Purpose of the Study:

• To develop a highly efficient CRISPR-Cas9 system for genetic manipulation in actinomycetes.
• To enable gene deletion, precise gene replacement, and reversible gene expression control.
• To facilitate systematic metabolic engineering of actinomycetes for secondary metabolite production.

Main Methods:

• Development and application of a CRISPR-Cas9 system targeting specific genes (actIORF1 and actVB) in Streptomyces coelicolor A3(2).
• Utilized homology-directed repair (HDR) and nonhomologous end joining (NHEJ) pathways for gene inactivation and deletion.
• Implemented a CRISPR interference (CRISPRi) system using a catalytically dead Cas9 (dCas9) for reversible gene expression control.

Main Results:

• The CRISPR-Cas9 system successfully inactivated targeted genes in Streptomyces coelicolor.
• NHEJ pathway resulted in variable-sized deletions when no HDR template was provided.
• Precise gene deletions were achieved with near 100% frequency when HDR templates were supplied.
• The CRISPRi system demonstrated efficient and reversible control of gene expression.

Conclusions:

• The developed CRISPR-Cas9 system is a powerful and broadly applicable tool for actinomycetal genome manipulation.
• This system overcomes previous limitations in genetic manipulation, facilitating metabolic engineering.
• It enables precise genetic modifications and gene expression control, paving the way for enhanced production of valuable compounds.