Biology subjects

Achache, W.

Publications and source records attributed to Achache, W..

3 recordsLinked to original sources

bioRxiv2025

Mycobacterial cell division arrest and smooth-to-rough envelope transition using CRISPRi-mediated genetic repression systems

The genetic basis underlying non-tuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well-established mycobacteria-optimized dCas9Sth1-mediated gene knockdown system to develop a new subset of fluorescence-based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use the model organism Mycobacterium smegmatis (M. smegmatis) as surrogate for NTM species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene-silencing when targeting essential genes but also genes involved in smooth-to-rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.

microbiology↗

bioRxiv2024

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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/616838v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@212460org.highwire.dtl.DTLVardef@1db5529org.highwire.dtl.DTLVardef@b426e2org.highwire.dtl.DTLVardef@164f628_HPS_FORMAT_FIGEXP M_FIG C_FIG Development and validation of a new subset of E. coli-Mycobacteria shuttle vectors that enable simultaneous CRISPRI-mediated gene silencing and fluorescence based single-cell imaging.

microbiology↗

bioRxiv2024

Emerging Mycobacterium bovis in Lebanon: a snapshot based on whole-genome sequencing.

BackgroundTuberculosis is a pressing public health issue in Lebanon, a country of approximately five million people, including around 1.5 million refugees from Palestine and Syria. Prior research has revealed uncontrolled animal sources of Mycobacterium bovis, emphasizing the necessity for a comprehensive approach to combat tuberculosis in the region. Methods48 clinical Mycobacterium tuberculosis complex isolates were identified through whole genome sequence. Also, 43 animal fecal samples were collected from various farms across Lebanon to investigate the presence of the M. tuberculosis complex using CRISPR-csm4 PCR. ResultsGenomic analysis revealed that 39/48 (81.25%) of isolates were M. tuberculosis and 9/48 (18.75%) were M. bovis. M. tuberculosis was distributed over four lineages, Indo-Oceanic L1 (n = 3/39)(7.6%), East-Asian L2 (n = 1/39)(2.5%), East-African Indian L3 (n = 5/39)(12.8%) and Euro-American L4 (n = 30/39)(76.9%). Sub-lineage L4.8 (Euro-American (mainly T), comprising 8/39 of the isolates (20.5%) was predominant, followed by sub-lineages L3 (East-African Indian, n = 5/39 isolates)(12.8%), L4.2.2.2 (Euro-American (Ural), n= 4/39 isolates)(10.2%) and L4.6.5 (Euro American, n=4/39 isolates)(10.2%). Nine M. bovis were classified into two clades, designated as unknown2 (n=2/9; 22.2%) and unknown3 (n=7/9; 77.8%). Interestingly, none of the clades or others were detected in the 48 faecal samples using CRISPR standard PCR and qPCR. ConclusionsThis study offers insights into human and bovine tuberculosis in Lebanon, emphasizing M. tuberculosis lineages prevalence and M. bovis distribution into two clades, aiding the fight against tuberculosis, especially bovine tuberculosis, and renewing our understanding of tuberculosis dynamics in Lebanon.

microbiology↗