Single and multiplexed gene repression in solventogenic Clostridium via Cas12a-based CRISPR interference

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• Clostridium species are valuable industrial microorganisms due to their metabolic capabilities but are challenging to genetically manipulate.
• Existing genetic tools in Clostridium are limited, hindering broader application and research.
• CRISPR interference (CRISPRi) offers a method for gene repression without DNA editing.

Purpose of the Study:

• To develop and validate a dCas12a-based CRISPR interference system for transcriptional gene repression in Clostridium species.
• To demonstrate the system's efficacy and applicability across different Clostridium species.
• To establish a foundation for high-throughput genetic screening in Clostridium.

Main Methods:

• Development of a dCas12a-based CRISPR interference system using the Francisella novicida Cas12a.
• Application of the system to target specific genes in Clostridium acetobutylicum and Clostridium pasteurianum.
• Demonstration of multiplexed gene repression using a single synthetic CRISPR array.

Main Results:

• Achieved >99% reduction in targeted gene transcript levels in Clostridium acetobutylicum.
• Achieved >75% reduction in targeted gene transcript levels in Clostridium pasteurianum.
• Demonstrated successful multiplexed repression and identified unique metabolic profiles associated with reduced gene expression.

Conclusions:

• The developed dCas12a-based CRISPRi system is effective for transcriptional gene repression in mesophilic Clostridium species.
• The system's broad applicability is attributed to the Francisella novicida Cas12a's suitability for low GC content DNA.
• This work provides a powerful tool for genetic manipulation and high-throughput screening in Clostridium, overcoming previous limitations.