Development of CRISPR-Cas3 for genome editing tool in Streptomyces

点击翻译按钮获取中文摘要

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.