Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/P_m expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.

中文翻译：

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用于可调控制恶臭假单胞菌基因表达的扩展 CRISPRi 工具箱。


由于其广泛的代谢多样性和生理稳健性，以及对基因操作的适应性和对应激条件的高抵抗力，恶臭假单胞菌日益成为工业和环境应用中一系列应用的首选生物体。然而，由于缺乏有效且高效地调控靶基因表达的特定遗传工具，一系列应用合成生物学和代谢工程方法仍然受到限制。在这里，我们提出了一种单质粒 CRISPR 干扰 (CRISPRi) 系统，该系统在诱导型 XylS/P _m表达系统的控制下表达核酸酶缺陷型cas9基因，并可选择采用组成型表达的引导 RNA（sgRNA 或 crRNA）和tracrRNA)。我们表明，该系统能够单独或同时对编码荧光蛋白的染色体表达基因进行可调节、严格控制的基因抑制（高达 90%）。此外，我们证明该方法可以抑制必需基因pyrF和ftsZ的表达，分别导致显着低的生长速率或形态变化。这种多功能系统扩展了当前 CRISPRi 工具箱的功能，可有效、有针对性和可控地操纵P基因表达。恶臭。