Building CRISPR Immunity Systems - How is Invading DNA Captured?

构建 CRISPR 免疫系统 - 如何捕获入侵 DNA？

• 批准号: BB/T006625/1
• 负责人: Edward Bolt
• 金额: $ 45.8万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT006625%2F1
• 关键词: Building; CRISPR; Immunity; Systems; How

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.

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可移动遗传元件(MGE)的多余遗传负担具有免疫力。当MGE DNA的片段被识别、捕获并存储在微生物的CRISPR系统中时，免疫系统就建立了--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被用来寻找和破坏返回的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的适应的过程的某些部分是已知的，然而，尚不清楚病毒DNA/RNA是如何被识别为“非我”的，因此在微生物第一次遇到它时被捕获以建立免疫力。我们知道Cas1-Cas2酶复合体对CRISPR适应是必不可少的，但我们并不完全知道如何在自然细胞系统中或在单个基因和蛋白质的分子细节中实现适应。我们将研究Cas1-Cas2酶复合体的细胞和分子生物学，以了解它如何捕获病毒DNA片段。这将使用大肠杆菌作为模式细菌，检查DNA捕获的生化，DNA捕获过程中至关重要的遗传成分，并使用活细胞的延时显微成像来实时和前所未有的详细观察适应情况。从微生物学和抗菌素耐药性、DNA断裂和基因组不稳定到基因工程的生物技术应用，有关CRISPR免疫如何在细菌中产生的新知识对许多不同的生物学领域都很重要。对于对抗生素耐药性感兴趣的微生物学家来说，理解免疫是如何产生的很重要，因为这是一个迫切需要克服的挑战。通过了解CRISPR在正常健康细菌中的免疫功能，将能够开发出自然的策略来克服抗生素耐药性，在这种情况下，耐药性基因通常携带在被CRISPR破坏的遗传元素上。我们在细胞中对Cas1进行成像的新方法也将有助于研究人员对了解细胞中的基因组动力学感兴趣，特别是DNA是如何以及为什么被破坏的。这与生物学家直接相关，他们希望了解基因组不稳定是如何产生的，并导致各种人类疾病(如癌症和衰老过程)中表现出的问题。CRISPR被广泛用作生物技术工具、遗传工程和细胞中的编辑，但Cas1-Cas2复合体不如其他基于CRISPR的基因编辑方法，如Cas9。了解Cas1-Cas2如何在将DNA分子存储到DNA片段数据库之前捕获DNA分子，例如CRISPR，有可能简化其在许多应用中作为编辑工具的使用。

项目成果

Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9.

• DOI: 10.1042/bsr20203595
• 发表时间: 2021-01-29
• 期刊: Bioscience reports
• 影响因子: 4
• 作者: Lau CH;Bolt EL
• 通讯作者: Bolt EL

Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing.

• DOI: 10.1093/nar/gkad032
• 发表时间: 2023-02-28
• 期刊: Nucleic acids research
• 影响因子: 14.9

CRISPR-Cas adaptation in Escherichia coli.

• DOI: 10.1042/bsr20221198
• 发表时间: 2023-03-31
• 期刊: Bioscience reports
• 影响因子: 4

A Tryptophan 'Gate' in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity.

• DOI: 10.3390/ijms22062848
• 发表时间: 2021-03-11
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: He L;Matošević ZJ;Mitić D;Markulin D;Killelea T;Matković M;Bertoša B;Ivančić-Baće I;Bolt EL
• 通讯作者: Bolt EL