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.

病毒是细胞生命的主要威胁之一。他们的目标是将遗传物质 DNA 或 RNA 注入宿主细胞，并利用宿主细胞的蛋白质机制进行自我繁殖。病毒不仅可以感染动物，还可以感染细菌。被称为噬菌体的细菌病毒是地球上最广泛和最多样化的遗传实体之一：例如，一茶匙海水可以含有超过 20 亿个噬菌体。为了保护自己免受这种病毒的攻击，细菌已经形成了多重防御屏障来抵御感染和感染。在这个项目中，我们的目标是了解一种这样的防御方法是如何工作的，即一种称为“CRISPR/Cas”的适应性微生物免疫系统。这些系统有两个主要作用：在外源 DNA 繁殖之前将其降解；并且，获取病毒遗传信息的短片段并将其整合到宿主基因组中。这赋予了感染记忆，这意味着如果再次感染，细胞可以更快地对该类型的病毒做出反应。我们想了解的是，CRISPR/Cas系统如何利用其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实现这一目标，这些技术统称为单分子酶学。基本原理是，我们将分离对该过程至关重要的蛋白质、DNA 和 RNA，然后使用特殊的显微镜，在其中我们可以单独测量单分子的活性。我们使用这些方法是因为我们正在研究的过程很复杂，如果我们使用装满分子的“试管”，我们将无法详细地解决该机制。我们将研究一种称为 Cas9 的蛋白质。当利用其核酸内切酶活性特异性靶向病毒 DNA 进行切割时，Cas9 使用源自宿主基因组中间隔元件线性阵列的 RNA 片段。这些间隔区代表先前病毒感染的基因片段。 Cas9-RNA 和目标 DNA 形成特定的碱基对，形成称为 R 环的混合结构。然而，这个 R 环是如何形成的仍然是个谜。我们的初步数据表明我们可以直接观察R环的形成。因此，我们可以测试这个过程并探索其机制。一个重要特征是称为“PAM”的 DNA 序列。这种 PAM 不存在于细菌基因组的 CRISPR 阵列中，但存在于病毒 DNA 上，因此可以区分“外来”和“自身”DNA。令人惊讶的是 PAM 序列非常简单；仅包含少数特定碱基。因此，我们还想了解 Cas9 如何决定切割位置。我们怀疑 Cas9 会快速扫描 DNA，从而扭曲 PAM 序列。当这种扭曲揭示出与 RNA 互补的部分序列时，就会形成 R 环。我们希望能够直接观察这一过程的发生，了解 Cas9 如何避免切割错误的 DNA。进行这项研究有趣且有价值的原因有很多。从根本上讲，这些实验将告诉我们这个过程是如何运作的。除了适应性免疫之外，Cas9 活性还让人想起同源重组中的序列搜索事件，同源重组是一个对于 DNA 修复非常重要的过程，当它崩溃时，会导致遗传疾病。最令人兴奋的是，目前人们对 CRISPR/Cas 系统作为“基因组手术”的潜在工具产生了巨大的兴趣。这种合成生物学技术为利用针对突变基因的酶直接修复细胞中的遗传病带来了希望。令人兴奋的是，Cas9 在人类细胞中的特异性基因靶向最近已得到证实。然而，由于该工具是一把切割 DNA 的分子剪刀，因此仅在正确的位置（人类 DNA 的 30 亿个碱基对中）进行切割至关重要。我们的研究将通过提供对 R 环如何以高度特异性的方式形成的更全面的理解来极大地帮助这一点。