Development of CRISPR-Cas3 for genome editing tool in Streptomyces

CRISPR Cas3与Cas9相似，只是Cas3既是核酸内切酶又是核酸外切酶。在最初的切割之后，外切酶产生大量的缺失，生物体必须修复这些缺失才能生存。所产生的缺失在长度上变化，上限至少为100 Mbp，并且可以发生在基因组中的几乎任何位置。这为全基因组诱变提供了重要的机会，特别是在没有有效转座子诱变系统的细菌中。链霉菌就是这种情况，它是我实验室研究项目中的主要细菌属。链霉菌因其“特殊代谢”、抗生素的重要来源、卓越的产孢程序和独特的“探索”运动模式而被广泛研究。第四个关键特征是它们与后生动物的相互作用。我们已经证明了链霉菌和线虫之间的第一次相互作用，确定了四类物种特异性相互作用。其中之一，一种强有力的化学吸引表型，是模式生物委内瑞拉链霉菌和其他四种野生分离株的特性。理解吸引力是至关重要的：这个领域最大的问题之一是这些互惠关系是如何建立的？为什么有些种类的链霉菌最终会与后生动物伴侣结合，而另一些则不会？目前，还没有对这些物种特异性选择的机制解释。为了解决这个问题，我们需要阐明秀丽隐杆线虫被委内瑞拉葡萄球菌吸引的遗传基础。要做到这一点，我们需要一个有效的全基因组诱变策略。为此，我们将通过以下三个目标开展Cas3诱变研究：1。我们将构建任何链霉菌的CRISPR-Cas3诱变载体，并表征其在委内瑞拉葡萄球菌中的作用。这将产生第一个基因组规模的链霉菌诱变工具。2. 在委内瑞拉葡萄染色体上创建全基因组缺失序列。我们将使用Cas3诱变来创建嵌套的缺失突变沿委内瑞拉s染色体的间隔。我们将描述已知链霉菌突变表型的突变体。这将在链霉菌研究的首映模型系统中创建第一个全基因组缺失突变体的集合。3. 确定吸引秀丽隐杆线虫的基因。我们将使用Cas3缺失来识别损害秀丽隐杆线虫吸引力的突变。我们将通过对这些突变体进行测序，并利用现有的Cas9系统进行靶向基因逐基因诱变来鉴定特定基因。这样，我们将阐明链霉菌和线虫之间的第一种吸引现象。这项工作将阐明链霉菌与后生动物之间的一种新的相互作用，并为链霉菌研究界提供一种急需的新的诱变工具。

CRISPR Cas3 is similar to Cas9 except that Cas3 is both endonuclease and exonuclease. After the initial cut, the exonuclease creates large deletions, which the organism must then repair to survive. The resulting deletions vary in length with an upper limit of at least 100 Mbp and can occur at virtually any location in a genome. This opens up important opportunities for genome-wide mutagenesis, particularly in bacteria for which there is no efficient transposon mutagenesis system. This is the case for Streptomyces, the principle bacterial genus in my lab's research programme. Streptomyces are widely studied for their `specialized metabolism', a critical source of antibiotics, their remarkable sporulation program and unique mode of 'exploration' motility. A fourth key trait is their mutualistic interactions with metazoan organisms. We have demonstrated the first interaction between streptomycetes and nematodes, identifying four classes of species-specific interactions. One of these, a potent chemical attraction phenotype, is a property of the model organism Streptomyces venezuelae and four other wild isolates. Understanding attraction is critically important: one of the biggest questions in the field is how these mutualistic relationships are established? Why do some species of Streptomyces end up associated with a metazoan partner while others do not? At present, there is no mechanistic explanation for these species-specific choices. To address this question we need to elucidate the genetic basis for attraction of C. elegans by S. venezulae. To do this we need an efficient genome-wide mutagenesis strategy. We will develop Cas3 mutagenesis to this end through the following three aims: 1. Develop CRISPR-Cas3 mutagenesis in streptomycetes We will construct vectors for CRISPR-Cas3 mutagenesis of any streptomycete and characterize their action in S. venezuelae. This will generate the first genome-scale mutagenesis tool for the streptomycetes. 2. Create a genome-wide deletion series in the S. venezuelae chromosome. We will use Cas3 mutagenesis to create nested deletion mutations at intervals along the S. venezuelae chromosome. We will characterize the resulting mutants for the known Streptomyces mutant phenotypes. This will create the first collection of genome-wide deletion mutants in the premiere model system for Streptomyces research. 3. Identify the gene(s) responsible for attracting C. elegans. We will use the Cas3 deletions to identify mutations that compromise the attraction of C. elegans. We will identify the specific genes by sequencing these mutants and targeted gene-by-gene mutagenesis with the existing Cas9 system. In this way we will elucidate the first attraction phenomenon between a streptomycete and a nematode. This work will illuminate a novel interaction between streptomycetes and metazoan organisms and provide a vitally needed new mutagenesis tool to the Streptomyces research community.