Fluorescence-based CRISPR interference system for controlled genetic repression and live single-cell imaging in mycobacteria

Mycobacterial genetics has played a pivotal role over the last 35 years in our understanding of mycobacterial physiology, pathogenesis and antibiotic resistance. Numerous approaches are now available worldwide to dissect the contribution of genes of interest in biological processes. However, many of these approaches can be fastidious, difficult to perform and time-consuming, especially when working with slow-growing mycobacteria or in bio-safety level two/three settings. The recent development of CRISPRi-mediated targeted gene repression has revolutionized the way research groups can perform genetics in mycobacteria, providing a fast, robust and efficient alternative to study the function of specific genes including essential genes. In this research letter, we report the development and validation of a new subset of fluorescence-based CRISPRi tools for our scientific community. The pJL series is directly derived from the original integrative pIRL2 and pIRL117 CRISPRi vectors and conserved all the elements required to perform inducible targeted gene repression. In addition, these vectors carry two distinct fluorescent markers for which the expression is driven by the strong and constitutive promotor psmyc to simplify the selection of recombinant clones. We demonstrate the functionality of these vectors by targeting the expression of the non-essential glycopeptidolipid translocase mmpL4b and the essential genes rpoB and mmpL3 . Finally, we describe an efficient single-step procedure to co-transform mycobacterial species with this integrative genetic tool alongside replicative vectors. Such tools and approaches should be useful to foster discovery in mycobacterial research.