CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Gene expression control is vital for protein production but complex due to multi-level regulation.
• Existing CRISPR tools face limitations in fine-tuning gene expression across diverse contexts.

Purpose of the Study:

• To optimize CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) targeting for the dCas9-α/ω system.
• To develop a novel CRISPR-assisted Oligonucleotide Annealing based Promoter Shuffling (OAPS) strategy to expand dCas9-ω applications.
• To systematically evaluate gene expression regulation factors and enhance protein production in Bacillus subtilis.

Main Methods:

• Defined optimal targeting windows for dCas9-α/ω CRISPRa and CRISPRi using position-specific guide RNAs (gRNAs).
• Developed and applied the CRISPR-assisted Oligonucleotide Annealing based Promoter Shuffling (OAPS) strategy for promoter library construction.
• Combined OAPS and dCas9-ω to investigate promoter effects, protein folding, and degradation on amylase BLA expression.

Main Results:

• Demonstrated the dCas9-α/ω system's capability to act as a master regulator for simultaneous gene activation and repression.
• The OAPS strategy successfully generated functional promoter mutants, facilitating library creation in low-efficiency transformation hosts.
• Achieved a 260-fold enhancement in amylase BLA production by integrating OAPS and dCas9-ω, optimizing multiple regulatory factors.

Conclusions:

• The dCas9-α/ω system offers precise, multi-directional, and multi-dimensional gene expression control in bacteria.
• The OAPS strategy significantly improves promoter engineering and library construction for microbial applications.
• This combined toolkit provides a powerful platform for optimizing protein production and synthetic biology endeavors.