Gene Silencing through CRISPR Interference in Mycoplasmas.

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• Mycoplasmas are pathogenic, genome-reduced bacteria crucial for systems and synthetic biology.
• Genetic manipulation in mycoplasmas is challenging, hindering research.
• Existing methods limit the study of essential genes and regulatory networks.

Purpose of the Study:

• To develop a versatile and efficient gene-editing tool for Mycoplasma.
• To enable functional genomics studies in genome-reduced bacteria.
• To facilitate the identification of novel therapeutic targets for Mycoplasma infections.

Main Methods:

• A single-plasmid transposon-based CRISPR interference (CRISPRi) system was engineered.
• Expression levels of dCas9 were optimized to avoid growth impairment.
• Proteomic responses to exogenous dCas9 expression were analyzed.
• The system was validated by targeting transcription factors (fur, spxA, whiA) and hup1 in Mycoplasma gallisepticum.
• Knockdown experiments were performed on metabolic genes in Mycoplasma hominis.

Main Results:

• The CRISPRi system effectively repressed target gene expression by tenfold in Mycoplasma gallisepticum.
• Gene repression influenced the mRNA levels of predicted target genes.
• The proteomic response to dCas9 expression in genome-reduced bacteria was characterized for the first time.
• The system demonstrated versatility by successfully knocking down metabolic genes in Mycoplasma hominis.

Conclusions:

• The developed CRISPRi system is a powerful tool for gene function discovery in Mycoplasma.
• This technology allows for deciphering complex regulatory networks in these bacteria.
• The system holds potential for identifying novel drug targets to combat Mycoplasma infections.