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

病毒是对细胞生命的主要威胁之一。他们的目标是将他们的遗传物质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。病毒不仅能感染动物，还能感染细菌。细菌病毒被称为噬菌体，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。为了保护自己免受病毒的攻击，细菌已经形成了多种防御感染的屏障，在这个项目中，我们的目标是了解其中一种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：在外来DNA可以繁殖之前将其降解；以及获取简短的病毒遗传信息并将其整合到宿主基因组中。这给人一种感染的记忆，这意味着如果细胞再次感染，它可以更快地对这种类型的病毒做出反应。我们想了解的是，CRISPR/CAS系统如何利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完成这项工作，这些技术统称为单分子酶学。其基本原理是，我们将分离对这一过程至关重要的蛋白质、DNA和RNA，然后使用特殊的显微镜，在其中我们可以测量分离出的单分子的活性。我们之所以使用这些方法，是因为我们正在研究的过程是复杂的，如果我们使用充满分子的“试管”，我们将无法详细地研究这种机制。我们将研究一种名为Cas9的蛋白质。当利用其核酸内切酶活性专门针对病毒DNA进行切割时，Cas9使用的是一段来自宿主基因组中间隔区元件线性阵列的RNA。这些间隔区代表以前病毒感染的基因片段。Cas9-RNA和目标DNA形成特定的碱基对，形成一种称为R-环的杂交结构。然而，这个R环是如何形成的还是个谜。我们的初步数据表明，我们可以直接观察到R环的形成。因此，我们可以测试这一过程，并探索其机制。一个重要的特征是被称为“PAM”的DNA序列。这种PAM不存在于细菌基因组上的CRISPR阵列中，而存在于病毒DNA中，因此它区分了“外来”和“自身”DNA。令人惊讶的是，PAM序列非常简单，只包含几个特定的碱基。因此，我们还想了解Cas9是如何决定削减哪里的。我们怀疑Cas9快速扫描DNA，在扫描过程中扭曲PAM序列。当这种扭曲揭示出与RNA互补的部分序列时，就形成了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。为什么进行这项研究是有趣和有价值的，有很多原因。从根本上讲，这些实验将教会我们这个过程是如何工作的。除了获得性免疫，Cas9的活性还让人想起同源重组中的序列搜索事件，这一过程对DNA修复非常重要，当它被破坏时，会导致遗传病。最令人兴奋的是，目前人们对CRISPR/CAS系统作为“基因组外科”的潜在工具有极大的兴趣。这项合成生物学技术提供了一种希望，即可以使用针对突变基因的酶直接在细胞中修复遗传病。令人兴奋的是，最近已经证实了Cas9在人类细胞中的特异性基因靶向。然而，由于这个工具是一把切割DNA的分子剪刀，因此它只切割正确的位置(在30亿个人类DNA碱基对中)是至关重要的。我们的研究将极大地帮助这一点，因为我们将提供更全面的理解，以高度特定的方式形成R环。

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.