Laudouze Janïs, Point Vanessa, Achache Wafaa, Crauste Céline, Canaan Stéphane, Santucci Pierre
Aix Marseille Univ, CNRS, LISM, IMM FR3479, IM2B, France.
IHU Méditerranée Infection, Aix-Marseille Univ., France.
FEBS Lett. 2025 Feb;599(4):488-501. doi: 10.1002/1873-3468.15071. Epub 2024 Dec 1.
In this research letter, we report the development and validation of a new subset of fluorescence-based CRISPR interference (CRISPRi) tools for our scientific community. The pJL series is directly derived from the original pIRL CRISPRi vectors and conserves all the elements to perform inducible targeted gene repression. These vectors carry two distinct fluorescent markers under the constitutive promoter psmyc to simplify the selection of recombinant clones. We demonstrate the functionality of these vectors by targeting the expression of the 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 episomal vectors. Such tools and approaches should be useful to foster discovery in mycobacterial research.
在这篇研究通讯中，我们报告了为科学界开发并验证的一组基于荧光的新型CRISPR干扰（CRISPRi）工具。pJL系列直接源自原始的pIRL CRISPRi载体，并保留了所有用于进行诱导型靶向基因抑制的元件。这些载体在组成型启动子psmyc下携带两种不同的荧光标记，以简化重组克隆的筛选。我们通过靶向糖肽脂转运酶mmpL4b以及必需基因rpoB和mmpL3的表达来证明这些载体的功能。最后，我们描述了一种高效的单步程序，可使用这种整合遗传工具与游离型载体共同转化分枝杆菌属物种。此类工具和方法应有助于促进分枝杆菌研究中的发现。
