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

病毒是细胞生命的主要威胁之一。他们的目标是将自己的遗传物质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复制。病毒不仅能感染动物，还能感染细菌。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，一茶匙海水可以包含超过20亿个噬菌体。为了保护自己免受这种病毒的攻击，细菌已经形成了多种防御感染的屏障，在这个项目中，我们的目标是了解这种防御方法是如何工作的，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它们整合到宿主基因组中。这就形成了对感染的记忆，这意味着如果病毒再次感染，细胞可以更快地对这种病毒做出反应。我们想了解的是，CRISPR/Cas系统如何利用它积累的遗传信息来针对特定的DNA序列。我们将使用最先进的跨学科技术，统称为单分子酶学。基本原理是，我们将分离出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，我们可以在其中测量分离的单分子的活性。我们使用这些方法是因为我们正在研究的过程是复杂的，如果我们使用充满分子的“试管”，我们将无法在任何地方解决机制的细节问题。我们将研究一种叫做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使用一段来自宿主基因组中间隔元件线性阵列的RNA。这些间隔代表了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的CRISPR阵列中，但存在于病毒DNA上，因此它可以区分“外来”DNA和“自身”DNA。令人惊讶的是，PAM序列非常简单；只包含几种特定碱基的。因此，我们也想了解Cas9如何决定在哪里切割。我们怀疑Cas9会快速扫描DNA，在扫描过程中扭曲PAM序列。当这种扭曲显示出与RNA互补的部分序列时，r环就形成了。我们希望能够直接观察这一过程，了解Cas9如何避免切割错误的DNA。进行这项研究之所以有趣和有价值，有很多原因。在基本层面上，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，Cas9的活性让人想起同源重组中的序列搜索事件，这是一个对DNA修复很重要的过程，当它被破坏时，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”的潜在工具有着极大的兴趣。这种合成生物学技术为利用靶向突变基因的酶在细胞中直接修复遗传疾病提供了希望。令人兴奋的是，Cas9在人类细胞中的特异性基因靶向最近得到了证实。然而，由于这个工具是一把分子剪刀，它只能在正确的地方切割DNA（在人类DNA的30亿个碱基对中），这是至关重要的。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，极大地帮助这一点。

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.