Wiechert Johanna, Badia Roigé Biel, Dohmen-Olma Doris, Zhang Xiafei, Stella Roberto G, Elliot Marie A, Frunzke Julia
Institute of Bio- and Geoscience-IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, North Rhine-Westphalia, Germany.
Department of Biology, McMaster University, Hamilton, Ontario, Canada.
mBio. 2025 Jul 9;16(7):e0038225. doi: 10.1128/mbio.00382-25. Epub 2025 May 28.
Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XSs) inhibiting expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. Interference by transcription factors can lead to counter-silencing of XS target promoters, but relief of this repression typically requires promoter engineering. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach by using nuclease-deficient dCas enzymes to counteract the Lsr2-like XS protein CgpS in or Lsr2 in . Systematic reporter studies with dCas9 and dCas12a and various guide RNAs revealed effective counter-silencing of different CgpS target promoters in response to guide RNA/dCas DNA binding, independent of promoter sequence modifications. The most prominent CRISPRcosi effect was observed when targeting the CgpS nucleation site, an effect that was also seen in when targeting a known Lsr2 nucleation site within the chloramphenicol biosynthesis gene cluster. Analyzing the system in strains lacking the XS protein CgpS revealed varying strengths of counteracting CRISPR interference effects based on the target position and strand. Genome-wide transcriptome profiling in single-guide RNA/dCas9 co-expressing wild-type strains revealed high counter-silencing specificity with minimal off-target effects. Thus, CRISPRcosi provides a promising strategy for the precise upregulation of XS target genes with significant potential for studying gene networks as well as for developing applications in biotechnology and synthetic biology.
Lsr2-like nucleoid-associated proteins act as xenogeneic silencers (XSs), repressing the expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. The targets of Lsr2-like proteins are very diverse, including prophage elements, virulence gene clusters, and biosynthetic gene clusters. Consequently, the targeted activation of XS target genes is of interest for fundamental research and biotechnological applications. Traditional methods for counter-silencing typically require promoter modifications. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach, utilizing nuclease-deficient dCas enzymes to counteract repression by Lsr2-like proteins in and . The strongest effect was observed when targeting the Lsr2 nucleation site. Genome-wide transcriptome profiling revealed high specificity with minimal off-target effects. Overall, CRISPRcosi emerges as a powerful tool for the precise induction of genes silenced by xenogeneic silencers, offering new opportunities for exploring gene networks and advancing biotechnological applications.
类Lsr2核仁相关蛋白作为异种沉默因子（XSs），抑制放线菌中水平获得的富含腺嘌呤 - 胸腺嘧啶的DNA的表达。转录因子的干扰可导致XS靶启动子的反沉默，但这种抑制的解除通常需要启动子工程改造。在本研究中，我们开发了一种新型的成簇规律间隔短回文重复序列（CRISPR）/dCas介导的反沉默（CRISPRcosi）方法，通过使用核酸酶缺陷型dCas酶来对抗嗜热栖热放线菌中的类Lsr2 XS蛋白CgpS或天蓝色链霉菌中的Lsr2。使用dCas9和dCas12a以及各种引导RNA进行的系统报告基因研究表明，响应引导RNA/dCas DNA结合，不同的CgpS靶启动子可实现有效的反沉默，而与启动子序列修饰无关。当靶向CgpS成核位点时观察到最显著的CRISPRcosi效应，在靶向氯霉素生物合成基因簇内已知的Lsr2成核位点时，在天蓝色链霉菌中也观察到了这种效应。在缺乏XS蛋白CgpS的嗜热栖热放线菌菌株中分析该系统，发现基于靶位置和链的反作用CRISPR干扰效应强度不同。在共表达单引导RNA/dCas9的嗜热栖热放线菌野生型菌株中进行全基因组转录组分析，结果显示具有高反沉默特异性且脱靶效应最小。因此，CRISPRcosi为精确上调XS靶基因提供了一种有前景的策略，在研究基因网络以及开发生物技术和合成生物学应用方面具有巨大潜力。
类Lsr2核仁相关蛋白作为异种沉默因子（XSs），抑制放线菌中水平获得的富含腺嘌呤 - 胸腺嘧啶的DNA的表达。类Lsr2蛋白的靶标非常多样，包括原噬菌体元件、毒力基因簇和生物合成基因簇。因此，XS靶基因的靶向激活对于基础研究和生物技术应用具有重要意义。传统的反沉默方法通常需要启动子修饰。在本研究中，我们开发了一种新型的成簇规律间隔短回文重复序列（CRISPR）/dCas介导的反沉默（CRISPRcosi）方法，利用核酸酶缺陷型dCas酶来对抗嗜热栖热放线菌和天蓝色链霉菌中类Lsr2蛋白的抑制作用。当靶向Lsr2成核位点时观察到最强的效应。全基因组转录组分析显示具有高特异性且脱靶效应最小。总体而言，CRISPRcosi成为精确诱导被异种沉默因子沉默的基因的有力工具，为探索基因网络和推进生物技术应用提供了新机会。
