CRISPR-based technologies have changed the way of performing bacterial genetics, helping to elucidate many aspects of genome editing. Researchers at Aix Marseille University in France have developed a new subset of fluorescence-based CRISPR interference (CRISPRi) tools. CRISPRi is an alternative to CRISPR-Cas-mediated genome editing. It is a genetic perturbation technique allowing for sequence-specific repression of gene expression. This technology has revolutionized mycobacterial genetics.
A whole-genome CRISPRi screen is a powerful tool to review functional genomics and dissect mycobacterial physiology and pathogenesis. Researchers developed a procedure to co-transform mycobacterial species with this integrative genetic tool. This simple single-step procedure allows co-transformation of CRISPRi-based vectors with episomal vectors (free, circular, extrachromosomal DNA) encoding alternative fluorophores. This method can enable rapid isolation of co-transformants in both fast- and slow-growing bacteria, including the non-pathogenic environmental strain Mycobacterium smegmatis and the tubercle bacilli, Mycobacterium tuberculosis.
The selection of recombinant clones remains fastidious in mycobacteria. By using genes encoding fluorescent proteins such as Green Fluorescent Protein (GFP), mWasabi (green) or dTomato (red), scientists can select the bacterial mutant strains easier. Cooking with green tomatos is not just for chefs anymore! By adding these fluorescence expression cassettes, recombinant clones can be selected easier and they can be imaged using live single-cell fluorescence microscopy for visually stunning images.
Hygromycin B from TOKU-E EU (H007) was used as a selection marker in this study. A simple bench-top imaging system can detect fluorescent recombinant clones from their non-fluorescent counterparts.
We look forward to supporting your research discoveries with these and other products for mycobacterial research.
Reference
Laudouze J et al (2025) Fluorescence-based CRISPR interference system for controlled genetic repression and live single-cell imaging in mycobacteria. FEBS Lett. Link