Analysis of the acquired microbial defence mechanism against foreign DNA by the CRISPR system

微生物的生命不断受到外来遗传因子的威胁，如质粒、转座子或质粒。最近，人们发现了一种新的防御系统，它赋予那些在外来DNA入侵中幸存下来的细胞遗传免疫力。该系统被称为CRISPR，由短间隔DNA序列的染色体阵列组成，这些序列源自外源遗传元件，由回文重复序列分开。一种类似siRNA的机制被认为可以解释对外源DNA的抗性，但这种防御机制的分子细节尚不清楚。本文分析了大肠杆菌CRISPR防御系统的基本步骤及其调控。杆菌建议实验分析CRISPR元件转录和加工成短crRNA，并研究几种Cas蛋白在该过程中的参与。我们的主要目标是了解crRNA依赖性外源DNA失活的分子机制。在我们的第一个结果中，我们可以识别新的启动子并表征其调控的几个要素。该提案的预期结果对使用微生物培养物的生物技术和食品工业具有直接重要性，并将具有非常有用的应用潜力，以防止抗生素耐药性的传播。

Microbial life is threatened constantly by the invasion of foreign genetic elements, such as phages, transposons or plasmids. Recently, a new defence system has been discovered, which confers inheritable immunity to cells that have survived the invasion of foreign DNA. The system, termed CRISPR, consists of chromosomal arrays of short spacer DNA sequences, originating from the foreign genetic elements, which are separated by palindromic repeats. A siRNA-like mechanism has been assumed to explain resistance to foreign DNA but the molecular details of this defence mechanism are not known. Here we propose the analysis of the basic steps of the CRISPR defence system and its regulation in E. coli. Experiments are suggested to analyze the transcription and processing of the CRISPR elements to short crRNAs and to study the involvement of several Cas proteins in this process. Our main objective is to understand the molecular mechanisms of the crRNA-dependent inactivation of foreign DNA. In our first results we could identify new promoters and characterize several elements of their regulation. The results expected from this proposal are of direct importance for biotechnology and food industries, which use microbial cultures and will have the very useful potential for applications against the spreading of antibiotic resistance.