Anti-plasmid and CRISPRi activity of a Pseudomonas oleovorans Type IV-A CRISPR-Cas system

IV-A 型 CRISPR-Cas 系统的食油假单胞菌的抗质粒和 CRISPRi 活性

• 批准号: 405858350
• 负责人: Professor Dr. Lennart Randau
• 金额: --
• 依托单位: Arbeitsgruppe Genetik
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/405858350?language=en
• 关键词: Anti; plasmid; CRISPRi; activity; Pseudomonas

Archaea and Bacteria have evolved a variety of strategies to cope with foreign nucleic acids. One of these strategies employs CRISPR-Cas systems, which were originally described to mediate adaptive prokaryotic immunity, providing defense against viral attacks. Cas proteins form ribonucleoprotein complexes with CRISPR RNAs (crRNAs). These RNA molecules contain so-called spacer sequences that identify target nucleic acids via base complementarity. Recently, it emerged that CRISPR-Cas systems can repurpose this RNA guidance mechanism as (i) the spacer content of CRISPR elements is highly dynamic and (ii) a large repertoire of divergent Cas proteins was observed. Our group studies two Class 1 CRISPR-Cas ribonucleoprotein complexes with reduced Cas protein content. First, we investigate the Type I-Fv complex of Shewanella putrefaciens, which was shown to target DNA in the absence of commonly observed large and small subunits. The crystal structure of this assembly suggests that unique features of the Cas proteins Cas5fv and Cas7fv compensate for the loss of these subunits. We aim to study the detailed DNA targeting mechanism of this complex and propose to analyze formation of crRNA-DNA interactions using biolayer interferometry and activity assays with Cas protein mutants and target DNA variants. Collaborative efforts aim to investigate the structural and functional involvement of the helicases Cas3 and DinG and the dynamics of DNA scanning. Second, we identified a Type IV CRISPR-Cas system in Aromatoleum aromaticum and produced and purified recombinant Type IV ribonucleoprotein complexes. Type IV CRISPR-Cas does not contain adaptation modules or known DNA nucleases and its cellular function is unknown. We aim to characterize the composition and stoichiometry of the Type IV complexes using gel-filtration and mass-spectrometry approaches. The apparent absence of DNA nucleases suggests that the unknown activity of these CRISPR-Cas systems might not rely on the degradation of target DNA. Therefore, we aim to study the formation and stability of Typ IV complex-mediated crRNA-DNA interactions, so-called R-loops, in vitro and in vivo using footprinting and bisulfite sequencing techniques. The co-occurrence of transposons in the vicinity of Type IV CRISPR-Cas systems indicates that they potentially facilitate crRNA-guided transposition. We aim to test this novel role of CRISPR-Cas systems by following transposon mobility in response to crRNA targeted R-loop formation using next generation sequencing.

古生菌和细菌已经进化出了多种应对外来核酸的策略。其中一种策略采用CRISPR-Cas系统，该系统最初被描述为介导适应性原核免疫，提供对病毒攻击的防御。Cas蛋白与CRISPR rna （crrna）形成核糖核蛋白复合物。这些RNA分子包含所谓的间隔序列，通过碱基互补来识别目标核酸。最近，人们发现CRISPR-Cas系统可以重新利用这种RNA引导机制，因为(i) CRISPR元件的间隔物含量是高度动态的，（ii）观察到大量不同的Cas蛋白。本课题组研究了两种Cas蛋白含量降低的1类CRISPR-Cas核糖核蛋白复合物。首先，我们研究了腐烂希瓦氏菌的I-Fv型复合体，该复合体在没有常见的大亚基和小亚基的情况下靶向DNA。这种组合的晶体结构表明，Cas5fv和Cas7fv蛋白的独特特征弥补了这些亚基的缺失。我们的目标是研究该复合物的详细DNA靶向机制，并建议使用生物层干涉法和Cas蛋白突变体和靶DNA变体的活性测定来分析crRNA-DNA相互作用的形成。合作的目的是研究解旋酶Cas3和DinG的结构和功能参与以及DNA扫描的动态。其次，我们在Aromatoleum aromaticum中鉴定了IV型CRISPR-Cas系统，并生产和纯化了重组IV型核糖核蛋白复合物。IV型CRISPR-Cas不含适应模块或已知的DNA核酸酶，其细胞功能尚不清楚。我们的目的是表征的组成和化学计量的IV型配合物使用凝胶过滤和质谱方法。DNA核酸酶的明显缺失表明，这些CRISPR-Cas系统的未知活性可能不依赖于靶DNA的降解。因此，我们的目的是研究IV型复合物介导的crRNA-DNA相互作用的形成和稳定性，即所谓的r环，在体外和体内使用足迹和亚硫酸盐测序技术。转座子在IV型CRISPR-Cas系统附近的共同出现表明它们可能促进crrna引导的转座。我们的目标是通过使用下一代测序来跟踪转座子迁移以响应crRNA靶向r环的形成，从而测试CRISPR-Cas系统的这种新作用。