Characterizing CRISPR-Cas systems with non-defensive functions

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，用于检测和根除外来遗传物质。然而，最近的研究强调了基因调控和免疫回避中的其他作用，这对CRISPR作为免疫系统的单一定义提出了挑战。目前尚不清楚CRISPR的内在作用以及它们如何影响原核生物的整体生理和行为。我们之前表明，最丰富的I型CRISPR-Cas系统可以通过破坏系统的内切酶Cas3很容易地转化为转录抑制因子。我们假设I型系统可以自然地执行类似的功能。通过在原核生物基因组中搜索基因组靶向CRISPR阵列，我们确定了植物病原菌白色黄单胞菌（Xanthomonas albilineans）是一个极有希望的案例。该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，共编码64个基因组靶向间隔序列。进一步分析表明，靶位点两侧有已知的原间隔邻近基序（PAMs）， I-C系统似乎有缺陷的Cas3。这些初步结果强烈表明，CRISPR-Cas系统在这种重要的农业细菌中可以作为转录抑制因子并调节一系列细胞过程。本提案的目标是表征X. albilineans中两种CRISPR-Cas系统的特性。我们假设这两个系统都通过缺乏Cas3活性来功能性表达和调节靶点的转录。为了验证这一假设，我们提出了以下两个目标：目标1：使用无细胞转录-翻译系统确定每个CRISPR-Cas系统的DNA靶向结果。目的2：确定各CRISPR-Cas系统在白胆足鼠中的表达模式和功能。如果成功，这项工作将揭示CRISPR-Cas系统超越适应性免疫的新作用，并为在原核生物世界中发现的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，该项目与SPP 2141的目标直接一致。

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.