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

IF 3.5 4区 生物学 Q1 Biochemistry, Genetics and Molecular Biology FEBS Letters Pub Date : 2024-12-01 DOI:10.1002/1873-3468.15071
Janïs Laudouze, Vanessa Point, Wafaa Achache, Céline Crauste, Stéphane Canaan, Pierre Santucci
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Abstract

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.

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分枝杆菌可控基因抑制和活单细胞成像的荧光CRISPR干扰系统。
在这封研究信中,我们为我们的科学界报告了基于荧光的CRISPR干扰(CRISPRi)工具的新子集的开发和验证。pJL系列直接衍生自原始的pIRL CRISPRi载体,并保留了执行诱导靶向基因抑制的所有元件。这些载体在组成启动子psmyc下携带两个不同的荧光标记,以简化重组克隆的选择。我们通过靶向糖肽类脂转位酶mmpL4b和必需基因rpoB和mmpL3的表达来证明这些载体的功能。最后,我们描述了一种有效的单步程序,用这种综合遗传工具和episomal载体共同转化分枝杆菌物种。这些工具和方法应该有助于促进分枝杆菌研究的发现。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
FEBS Letters
FEBS Letters 生物-生化与分子生物学
CiteScore
7.00
自引率
2.90%
发文量
303
审稿时长
1.0 months
期刊介绍: FEBS Letters is one of the world''s leading journals in molecular biology and is renowned both for its quality of content and speed of production. Bringing together the most important developments in the molecular biosciences, FEBS Letters provides an international forum for Minireviews, Research Letters and Hypotheses that merit urgent publication.
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