Characterizing CRISPR-Cas systems with non-defensive functions

表征具有非防御功能的 CRISPR-Cas 系统

• 批准号: 405891106
• 负责人: Professor Dr. Chase Beisel
• 金额: --
• 依托单位: Helmholtz-Institut für RNA-basierte Infektionsforschung (HIRI)
• 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/405891106?language=en
• 关键词: Characterizing; CRISPR; Cas; systems; non

CRISPR-Cas systems are widely recognized as RNA-guided, adaptive immune systems in prokaryotes that detect and eradicate foreign genetic material. However, recent work highlighting alternative roles in gene regulation and immune avoidance are challenging the singular definition of CRISPR as an immune system. Still unclear are the inherent roles of CRISPR and how they impact the overall physiology and behavior of prokaryotic life.We previously showed that the most abundant Type I CRISPR-Cas systems can be readily converted into transcriptional repressors by disrupting the system’s endonuclease Cas3. We hypothesized that Type I systems may perform a similar function naturally. By searching prokaryotic genomes for genome-targeting CRISPR arrays, we identified the plant pathogen Xanthomonas albilineans as an extremely promising case. The genome of this bacterium contains two complete Type I CRISPR-Cas systems, a I-C system and a I-F system, encoding a total of 64 genome-targeting spacers. Further analysis showed that the target sites are flanked by known protospacer-adjacent motifs (PAMs), and the I-C system appears to have a defective Cas3. These preliminary results strongly suggest that the CRISPR-Cas systems in this agriculturally important bacterium could act as transcriptional repressors and regulate a collection of cellular processes.The goal of this proposal is to characterize the properties of the two CRISPR-Cas systems in X. albilineans. We hypothesize that both systems are functionally expressed and regulate transcription of target sites through lack of Cas3 activity. To test this hypothesis, we propose the following two objectives: Objective 1: Determine the outcome of DNA targeting by each CRISPR-Cas system using cell-free transcription-translation systems.Objective 2: Determine the expression pattern and function of each CRISPR-Cas system in X. albilineans. If successful, the proposed work could reveal novel roles of CRISPR-Cas systems that extend beyond adaptive immunity and provide the basis to identify similar functions across the diversity of Type I CRISPR-Cas systems found in the prokaryotic world. The PI has extensive experience with CRISPR-Cas systems and bacterial genetics, and the project directly aligns with the goals of SPP 2141.

CRISPR-Cas系统被广泛认为是原核生物中RNA引导的适应性免疫系统，可以检测和消除外源遗传物质。然而，最近的研究强调了CRISPR在基因调控和免疫回避中的替代作用，这对CRISPR作为免疫系统的单一定义提出了挑战。CRISPR的内在作用以及它们如何影响原核生物的整体生理学和行为仍然不清楚。我们以前表明，最丰富的I型CRISPR-Cas系统可以通过破坏系统的内切核酸酶Cas 3容易地转化为转录阻遏物。我们假设I型系统可能会自然地执行类似的功能。通过在原核基因组中搜索基因组靶向CRISPR阵列，我们将植物病原体黄单胞菌确定为一个非常有希望的案例。这种细菌的基因组包含两个完整的I型CRISPR-Cas系统，一个I-C系统和一个I-F系统，编码总共64个基因组靶向间隔区。进一步的分析表明，靶位点的侧翼是已知的前间区序列邻近基序（PAM），并且I-C系统似乎具有缺陷的Cas 3。这些初步结果强烈表明，CRISPR-Cas系统在这种农业上重要的细菌可以作为转录阻遏物和调节细胞过程的集合。阿尔比利尼人我们假设这两个系统都是功能性表达的，并且通过缺乏Cas 3活性来调节靶位点的转录。为了验证这一假设，我们提出了以下两个目标：目标1：确定每个CRISPR-Cas系统使用无细胞转录-翻译系统进行DNA靶向的结果目标2：确定每个CRISPR-Cas系统在X中的表达模式和功能。阿尔比利尼人如果成功的话，这项工作可以揭示CRISPR-Cas系统的新作用，这些作用超越了适应性免疫，并为在原核世界中发现的I型CRISPR-Cas系统的多样性中识别类似功能提供了基础。PI在CRISPR-Cas系统和细菌遗传学方面拥有丰富的经验，该项目直接符合SPP 2141的目标。