Building CRISPR Immunity Systems - How is Invading DNA Captured?

CRISPR系统已经在微生物中进化，使它们对死亡或来自病毒和其他移动的遗传元件（MGE）的不想要的遗传行李具有免疫力。当MGE DNA片段被识别、捕获并储存在微生物的CRISPR系统中时，免疫系统就建立起来了--这些过程被称为“适应”。一旦储存，CRISPR中的MGE DNA片段通过转录转化为RNA，CRISPR RNA用于寻找和破坏返回的MGE DNA，从而保护微生物细胞免受再次感染和死亡。 控制基于CRISPR的适应过程的某些部分是已知的，但是病毒DNA/RNA如何被识别为“非自身”并因此在微生物第一次遇到时被捕获以建立免疫力尚不清楚。我们知道Cas 1-Cas 2酶复合物对于CRISPR适应是必不可少的，但我们不完全知道在天然细胞系统中或单个基因和蛋白质的分子细节中如何实现适应。我们将研究Cas 1-Cas 2酶复合物的细胞和分子生物学，以了解它如何捕获病毒DNA片段。这将使用E。大肠杆菌作为一种模式细菌，检查DNA捕获的生物化学，遗传成分是这个过程的重要组成部分，并使用活细胞的延时显微成像来观察适应在真实的时间和前所未有的细节。关于CRISPR免疫力如何在细菌中发展的新知识对于许多不同的生物学领域都很重要，从微生物学和抗菌素耐药性，DNA断裂和基因组不稳定性到基因工程的生物技术应用。了解细菌中如何产生免疫力对于对抗生素耐药性感兴趣的微生物学家来说非常重要，因为这是一个迫切需要克服的挑战。通过了解CRISPR免疫在正常健康细菌中的功能，将有助于开发克服抗生素耐药性的天然策略，其中耐药性基因通常携带在被CRISPR破坏的遗传元件上。我们用于细胞中Cas 1成像的新方法也将使有兴趣了解细胞中基因组动态的研究人员受益，特别是DNA如何以及为什么会被破坏。这与希望了解基因组不稳定性如何产生并导致各种人类疾病（如癌症和衰老过程）中出现的问题的生物学家直接相关。 CRISPR被广泛用作生物技术工具基因工程和细胞编辑，但Cas 1-Cas 2复合物的开发不如其他基于CRISPR的基因编辑方法，如Cas9。了解Cas 1-Cas 2如何在将其存储在DNA片段数据库（例如CRISPR）中之前捕获DNA分子，有可能简化其在许多应用中作为编辑工具的使用。

CRISPR systems have evolved in microbes to give them immunity against death or unwanted genetic baggage from viruses and other mobile genetic elements (MGEs). The immunity system is built when fragments of MGE DNA are recognised, captured and stored in the microbe's CRISPR system - these processes are called "adaptation". Once stored, the MGE DNA fragments in CRISPR are converted into RNA by transcription, and the CRISPR RNA is used to seek and destroy returning MGE DNA, therefore protecting the microbial cell from re-infection and death. Some parts of the processes that control CRISPR-based adaptation are known, however it is unknown how viral DNA/RNA is recognised as "non-self" and is therefore captured to establish immunity the first time it is encountered by the microbe. We know that Cas1-Cas2 enzyme complex is essential for CRISPR adaptation, but we do not know fully how adaptation is achieved either in natural cellular systems or in the molecular detail of individual genes and proteins. We will investigate the cell and molecular biology of the Cas1-Cas2 enzyme complex to understand how it can capture fragments of virus DNA. This will be performed using E. coli as a model bacterium, examining the biochemistry of DNA capture, the genetic components that are vital parts of the process and using time lapse microscopic imaging of live cells to observe adaptation in real time and in unprecedented detail. The new knowledge of how CRISPR immunity develops in bacteria is important for many different areas of biology, from microbiology and antibacterial resistance, DNA breaks and genome instability to the biotechnology applications of genetic engineering.Understanding how immunity is generated in bacteria is important for microbiologists who are interested in antibiotic resistance as this is a challenge that urgently needs to be overcome. By knowing how CRISPR immunity functions in normal healthy bacteria will enable the development of natural strategies to overcome antibiotic resistance where the resistance genes are often carried on genetic elements that are destroyed by CRISPR.Our new methods for imaging of Cas1 in cells will also benefit researchers interested in understanding genome dynamics in cells, specifically how and why DNA gets broken. This is directly relevant to biologists who wish to understand how genome instability arises and leads to the problems manifested in various human diseases such as cancer, and the ageing process. CRISPR is widely used as biotechnology tool genetic engineering and editing in cells, but the Cas1-Cas2 complex is not as well developed as other CRISPR-based genetic editing methods e.g. Cas9. Understanding how Cas1-Cas2 can capture DNA molecules before storing them in a DNA fragment database e.g. CRISPR has potential to streamline its use as an editing tool in many applications.