CRISPR/Cas-directed transposition in Tn7-like elements

项目摘要/摘要 我们对导致收购的分子机制感兴趣 细菌的致病机制、功能和耐药性。我们正在调查一名 基因组在一个被称为转座的过程中改变的方式。转座子是 可在细胞内移动的可移动DNA元件，几乎存在于每种类型的 并在人类基因组中大量存在。 我们正在研究的特定转座子也具有重要的实际应用 在细菌中使用CRISPR/Cas进行基因组修改(可能还会更远)。 CRISPR-CAS系统正在革命性地改变我们对广泛的 有机体。然而，当这些系统出现时，它受到一个根本的限制 到插入新的遗传信息；这些系统在DNA中进行精确的断裂， 而是依靠内源性宿主修复机制来插入所需的新序列 信息(第二条DNA链同时转化到细胞中)。CRISPR- CAS系统天然存在于细菌和古菌中，以保护其免受病毒侵袭。 虽然所有CRISPR-CAS系统都利用引导RNA来靶向销毁病毒，但它们 是极其多样化的。我们的初步工作表明存在一个新的最小 转座子中功能异常的CRISPR-Cas系统。 在这项拨款中，我们将在大肠杆菌中建立CRISPR/Cas转座子靶向系统， 一种很容易在实验室中操纵以研究其基本功能的细菌。我们 还将修改该系统，使其能在不同类型的细菌中工作。我们将学习基本的 系统的功能并分离突变组件，这将使我们能够 系统更高效、更具体。 与公共健康的相关性：我们将确定允许 新出现的病原体和多重耐药细菌的进化 遗传信息的转移。这将导致更好的治疗方法和策略来限制 更严重病原体的进化。公共卫生也将得到服务，因为我们 将开发一项重要的新基因组修改技术，该技术将广泛应用于 适用于细菌，也可能适用于其他种类的生物体。

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.