CRISPR/Cas-directed transposition in Tn7-like elements

项目概要/摘要 我们感兴趣的分子机制，导致收购 致病功能和细菌的抗生素耐药性。我们正在调查 基因组在一个叫做转座的过程中被改变的方式。转座子是 移动的DNA元件，可以在细胞内移动，几乎在每种类型的 在人类基因组中含量丰富。 我们正在研究的特定转座子也有重要的实际应用 在细菌中使用CRISPR/Cas进行基因组修饰（以及潜在的）。 CRISPR-Cas系统正在革命性地改变我们对各种各样的基因进行遗传修饰的能力。 有机体然而，这些系统受到一个基本的限制， 插入新的遗传信息;这些系统在DNA中精确断裂， 而是依赖于内源性宿主修复机制来插入所需的新序列 信息（同时转化到细胞中的第二条DNA链）。CRISPR- Cas系统天然存在于细菌和古细菌中，用于保护免受病毒侵害。 虽然所有CRISPR-Cas系统都利用向导RNA来靶向病毒以进行破坏，但它们 是非常多样化的。我们的初步工作表明，存在一种新的最小 CRISPR-Cas系统在转座子中的不寻常功能。 在这项资助中，我们将在大肠杆菌中建立CRISPR/Cas转座子靶向系统。大肠杆菌， 这是一种很容易在实验室中操作的细菌，可以研究其基本功能。我们 也将修改系统，使其在不同类型的细菌中工作。我们将学习基本的 系统的功能和分离突变的组件，这将使我们能够使 系统更高效，更具体。 与公共卫生的相关性：我们将确定允许 新出现的病原体和多重耐药细菌的演变， 遗传信息的转移。这将导致更好的治疗和策略，以限制 更严重的病原体的进化公共卫生也将得到服务，因为我们 将开发一种重要的新基因组修改技术， 适用于细菌和可能的其他种类的生物体。

Project Summary / Abstract We are interested in the molecular mechanisms that lead to the acquisition of pathogenesis functions and antibiotic resistance in bacteria. We are investigating one of the ways genomes can be altered in a process called transposition. Transposons are mobile DNA elements that can move within a cell and are found in nearly every type of organism and are abundant in the human genome. The specific transposon we are working with also has important practical applications for genome modification using CRISPR/Cas in bacteria (and potentially beyond). CRISPR-Cas systems are revolutionizing our ability to genetically modify a wide array of organisms. However, these systems suffer from a fundamental limitation when it comes to inserting new genetic information; these systems make a precise break in the DNA, but rely on endogenous host repair mechanisms to insert the desired new sequence information (a second DNA strand transformed into the cell at the same time). CRISPR- Cas systems are found naturally in bacteria and archaea for protection from viruses. While all CRISPR-Cas systems utilize a guide RNA to target a virus for destruction they are extremely diverse. Our preliminary work indicated the existence of a novel minimal CRISPR-Cas system of unusual function in transposons. In this grant, we will establish the CRISPR/Cas transposon targeting system in E. coli, a type of bacteria that is easy to manipulate in the lab to study its basic functioning. We will also modify the system to work in different types of bacteria. We will study the basic functioning of the system and isolate mutant components that will allow us to make the system more efficient and more specific. Relevance to Public Health: We will determine the molecular mechanisms that allow the evolution of emerging pathogens and multi drug resistant bacteria though the transfer of genetic information. This will lead to better treatments and strategies to limit the evolution of more serious pathogens. Public health will also be served because we will be developing an important new genome modification technique that will be broadly applicable in bacteria and possibly other kinds of organisms.