Analysis of CRISPR-Cas systems in Cyanobacteria

蓝藻中 CRISPR-Cas 系统的分析

• 批准号: 206949509
• 负责人: Professor Dr. Wolfgang R. Hess
• 金额: --
• 依托单位: Institut für Biologie III
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/206949509?language=en
• 关键词: Analysis; CRISPR; Cas; systems; Cyanobacteria

In the here applied funding period we will continue our work to elucidate the three CRISPR-Cas systems (one is I-D, one is an atypical III and one is III-B subtype) in the cyanobacterium Synechocystis 6803. Following FLAG-tagging and immunoprecipitation, the exact composition of RNP complexes will be determined. Mutant phenotypes will be characterized and complementation studies be performed. We will introduce a synthetic CRISPR locus on the conjugative pVZ vector which we will use to investigate prerequisites for spacer recognition and repeat characteristics. We will go further into the structural analysis of bacterial I-D proteins, the subtype about which the least information is available at the current time. The transcription of CRISPR-Cas genes and cassettes in Synechocystis 6803 is controlled by regulatory proteins that have recently been identified by us and we plan to identify the signaling components leading to this regulation and to elucidate the physiological role of it. Despite some search efforts, there is still no bacteriophage known infecting Synechocystis 6803. However, in strain 6714, that is closely related to strain 6803, we have identified a 22.5 kb prophage insertion that can be induced. In addition, we will continue to search for native bacteriophages infecting either of these Synechocystis strains and will extend our analyses on further suitable cyanobacterial strains, including a more detailed analysis of the CRISPR-Cas systems of Synechocystis 6714 (one is an atypical I-A or I-B and two distinct III-B subtypes). In the last section of our project we will analyze samples from pilot plant facilities in which the large-scale cultivation of cyanobacteria engineered for the production of 3rd generation biofuels and other beneficial metabolites takes place with cultivation times of up to several months. Thus, there is an extreme scaling factor compared to traditional biotechnology in these facilities and the risk of infections and cultivation collapsing due to phage contamination is high and consequently, the challenges for the endogenous CRISPR systems substantial. Here we will investigate how are the CRISPR-based immune systems of such production strains evolving under these conditions? This project part will shed light on the CRISPR microevolution and is relevant for the practical application of the obtained knowledge.

在此申请的资助期间，我们将继续我们的工作，以阐明蓝藻集胞藻6803中的三个CRISPR-Cas系统（一个是I-D，一个是非典型III，一个是III-B亚型）。FLAG标记和免疫沉淀后，将确定RNP复合物的确切组成。将表征突变表型并进行互补研究。我们将在接合性pVZ载体上引入合成的CRISPR基因座，我们将使用该基因座来研究间隔区识别和重复特征的先决条件。我们将进一步研究细菌I-D蛋白的结构分析，这是目前信息最少的亚型。集胞藻6803中CRISPR-Cas基因和盒的转录受我们最近鉴定的调节蛋白的控制，我们计划鉴定导致这种调节的信号传导组分并阐明其生理作用。尽管进行了一些搜索努力，仍然没有已知的噬菌体感染集胞藻6803。然而，在与菌株6803密切相关的菌株6714中，我们已经鉴定了可以诱导的22.5kb前噬菌体插入。此外，我们将继续寻找感染这些集胞藻菌株中任一种的天然噬菌体，并将我们的分析扩展到其他合适的蓝藻菌株，包括对集胞藻6714的CRISPR-Cas系统的更详细分析（一种是非典型的I-A或I-B和两种不同的III-B亚型）。在我们项目的最后一部分，我们将分析来自中试工厂设施的样品，其中大规模培养蓝藻，用于生产第三代生物燃料和其他有益代谢产物，培养时间长达数月。因此，与这些设施中的传统生物技术相比，存在极端的缩放因子，并且由于噬菌体污染而导致的感染和培养崩溃的风险很高，因此，内源性CRISPR系统的挑战很大。在这里，我们将研究这些生产菌株的基于CRISPR的免疫系统在这些条件下是如何进化的？该项目部分将阐明CRISPR微进化，并与所获得知识的实际应用相关。