Understanding the pathways to R-loop formation by CRISPR/Cas immunity endonucleases

了解 CRISPR/Cas 免疫核酸内切酶形成 R 环的途径

基本信息

批准号：
BB/L000873/1
负责人：
Mark Dominik Szczelkun
金额：
$ 43.23万
依托单位：
University of Bristol
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2014
资助国家：
英国
起止时间：
2014 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL000873%2F1
关键词：
Understanding pathways loop formation CRISPR

项目摘要

Viruses are one of the major threats to cellular life. Their goal is to inject their genetic material, DNA or RNA, into a host cell and to reproduce themselves using the host cell's protein machinery. Viruses not only infect animals, they can also infect bacteria. The bacterial viruses, called bacteriophage, are amongst the most widespread and diverse genetic entities on Earth: for example, one teaspoon of seawater can contain over 2 billion bacteriophage.To protect themselves against this viral onslaught, bacteria have developed multiple defence barriers against infection and in this project we aim to understand how one such defence method works, an adaptive microbial immune system called "CRISPR/Cas". These systems have two main roles: To degrade foreign DNA before it can reproduce; and, to acquire short pieces of viral genetic information and to incorporate them into the host genome. This gives a memory of infection that means the cell can more quickly respond to that type of virus if it re-infects. What we want to learn is, how does the CRISPR/Cas system use the genetic information it accumulates to target specific DNA sequences. We will do this using state-of-the-art, interdisciplinary techniques that are collectively called single-molecule enzymology. The underlying principle is that we will isolate the proteins, DNA and RNA critical for the process and then use special microscopes in which we can measure the activities of single-molecules in isolation. We are using these approaches because the processes we are studying are complex, and if we were to use "test tubes" full of molecules we would not be able to address the mechanism in anywhere near as much detail.We will study a protein called Cas9. When specifically targeting viral DNA for cleavage using its endonuclease activity, Cas9 uses a piece of RNA that is derived from a linear array of spacer elements in the host genome. These spacers represent gene fragments from previous viral infections. The Cas9-RNA and target DNA make specific base pairs to form a hybrid structure called an R-loop. However, it is a mystery how this R-loop forms. Our preliminary data shows that we can directly observe R-loop formation. We can therefore test this process and explore its mechanism. An important feature is a DNA sequence called the "PAM". This PAM is not present in the CRISPR array on the bacterial genome but is present on the viral DNA, and it thus distinguishes "foreign" from "self" DNA. Surprisingly the PAM sequence is very simple; comprising only a few specific bases. We therefore also want to understand how Cas9 decides where to cut. We suspect that Cas9 rapidly scans the DNA, distorting PAM sequences as it goes. Where this distortion reveals a partial sequence complementary to the RNA, an R-loop forms. We want to be able to directly watch this process happening, to learn how Cas9 avoids cutting the wrong DNA.There are many reasons why it is interesting and valuable to undertake this study. At a fundamental level these experiments will teach us how this process works. Beyond adaptive immunity, Cas9 activity is reminiscent of sequence searching events in homologous recombination, a process that is important for DNA repair and which, when it breaks down, leads to genetic disease. Most exiting of all, there is currently enormous interest in CRISPR/Cas systems as potential tools for "genome surgery". This synthetic biology technique offers the hope that genetic disease could be directly repaired in cells using enzymes that target mutant genes. Excitingly, specific gene-targeting by Cas9 in human cells has recently been demonstrated. However, since this tool is a pair of molecular scissors that cuts DNA, it is vital that it only cuts in the correct place (amongst three billion base pairs of human DNA). Our study will greatly assist this by providing a much fuller understanding of how the R-loop is formed in a highly-specific manner.

病毒是对细胞生活的主要威胁之一。他们的目标是将其遗传物质DNA或RNA注入宿主细胞中，并使用宿主细胞的蛋白质机械重现自己。病毒不仅感染动物，还可以感染细菌。 The bacterial viruses, called bacteriophage, are amongst the most widespread and diverse genetic entities on Earth: for example, one teaspoon of seawater can contain over 2 billion bacteriophage.To protect themselves against this viral onslaught, bacteria have developed multiple defence barriers against infection and in this project we aim to understand how one such defence method works, an adaptive microbial immune system called "CRISPR/Cas".这些系统具有两个主要角色：在外国DNA可以繁殖之前降解；并且，要获取简短的病毒遗传信息并将其纳入宿主基因组。这给人以感染的记忆，这意味着如果细胞重新感染了该类型的病毒。我们要学习的是，CRISPR/CAS系统如何使用它积累的遗传信息来靶向特定的DNA序列。我们将使用最先进的跨学科技术来做到这一点，这些技术统称为单分子酶学。基本原理是我们将分离蛋白质，DNA和RNA对于该过程至关重要，然后使用特殊的显微镜，在该显微镜中我们可以分别测量单分子的活性。我们之所以使用这些方法，是因为我们正在研究的过程很复杂，如果我们要使用装满分子的“测试管”，我们将无法在附近的任何细节附近解决该机制。我们将研究一种称为Cas9的蛋白质。当使用其核酸内切酶活性的特定靶向病毒DNA进行裂解时，CAS9使用的是源自宿主基因组中隔离元件的线性序列阵列的RNA。这些间隔者代表了先前病毒感染的基因片段。 CAS9-RNA和靶DNA使特定的碱基对形成称为R环的杂种结构。但是，这是一个谜，这是一个谜。我们的初步数据表明，我们可以直接观察R环的形成。因此，我们可以测试此过程并探索其机制。一个重要的特征是称为“ PAM”的DNA序列。这种PAM不存在于细菌基因组上的CRISPR阵列中，而是在病毒DNA上存在，因此将“外来”与“自我” DNA区分开。令人惊讶的是，PAM序列非常简单。仅包括少数特定基础。因此，我们还想了解CAS9如何决定削减哪里。我们怀疑CAS9迅速扫描DNA，会随着它的形式扭曲PAM序列。如果这种失真揭示了与RNA互补的部分序列，则形成了R环。我们希望能够直接观察这一过程，以了解CAS9如何避免切割错误的DNA。有很多原因为什么进行这项研究很有趣和有价值。在基本层面上，这些实验将教我们这个过程的工作方式。除了自适应免疫之外，CAS9活性让人联想到同源重组中的序列搜索事件，这对于DNA修复很重要，并且在分解时会导致遗传疾病。目前，大多数退出的人都对CRISPR/CAS系统具有巨大的兴趣，作为“基因组手术”的潜在工具。这种合成生物学技术提供了希望，可以使用靶向突变基因的酶在细胞中直接修复遗传疾病。令人兴奋的是，最近已经证明了CAS9在人类细胞中的特定基因靶向。但是，由于该工具是一对切割DNA的分子剪刀，因此至关重要的是，它仅在正确的位置切割（在30亿个碱基对人的DNA中）至关重要。我们的研究将通过更深入地了解如何以高度特定方式形成R-loop来帮助这一点。