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

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.

The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system in bacteria and archaea. Silencing of foreign nucleic acids by the Type II CRISPR3/Cas system of Streptococcus thermophilus relies on a complex between the Cas9 nuclease and a small CRISPR RNA (crRNA), the latter derived by processing transcribed CRISPR repeat-spacer arrays. Base-pairing between the crRNA and a viral protospacer sequence forms an R-loop that activates the nuclease. A Protospacer-Adjacent Motif (PAM) is also required for R-loop formation. Despite analysis of several CRISPR/Cas systems, R-loop formation is not well understood. Even more puzzling is that PAMs comprise only a few specific nucleotides, suggesting that Cas9 must have to interrogate many PAMs before finding a protospacer. We aim to address these problems using single-molecule assays. We will first measure the fundamental thermodynamics of R-loop formation using a magnetic tweezers microscope by following DNA length changes as a function of torque. We will then establish the influence of the PAM, crRNA and protospacer in R-loop initiation. These studies will be backed-up using FRET measurements in a TIRF microscope (TIRFM), where we can follow changes in conformation between DNA, RNA and protein. We will also use the TIRFM to probe the DNA structure as Cas9 interacts with a PAM, to test if distortion seeds the R-loop, possibly due to transient DNA unwinding. Finally, we will fluorescently-label the Cas9-crRNA complex to directly follow its movement along a stretched DNA in the TIRFM. This will establish whether Cas9 uses 1-D and/or 3-D pathways during homology search and how this is influenced by interactions with orphan PAMs. This work will explain how CRISPR/Cas systems achieve specificity during RNA-guided immunity and will be particularly important in the manipulation of Cas9 as a tool for genome surgery, where specificity is paramount.

Academic researchers in the field of synthetic biology will benefit from this project. There are a great many labs striving to develop new DNA cleavage tools for genome surgery. The potential health benefits of safe, targeted genome therapy are huge, with accompanying benefits for the nation's economy. The CRISPR/Cas endonucleases have very recently been demonstrated to provide another system from which such tools can be developed. In order to exploit these enzymes safely, we must understand how the homology search process works and how cleavage at non-specific sites can be avoided. These are precisely the questions being sought in the basic research described in this project.

Biotechnology firms who are interested in developing gene therapy will benefit from this project. Companies such as Sangomo and Cellectis are investing heavily in zinc-finger nucleases and TALENs as genome cleavage tools. The CRISPR/Cas immunity endonucleases offer the possibility of new and readily programmable nucleases. This project seeks to understand how these enzymes recognise sequences in a specific manner, and will thus increase a knowledge base that such companies could use to improve and develop their products.

The PDRA employed on this project will obtain training in state-of-the-art single molecule methods. This will give them a distinct technical edge in their future careers and will add to the UK's infrastructure in workers capable of applying interdisciplinary techniques. The project will also add value to the research-led environment for training undergraduate students at the University of Bristol, who will be able to use the skills in quantitative analysis that they acquire in a wide-range of other occupations.

This project also fosters an international collaboration between researchers in three EU countries: the UK, Germany and Lithuania. There will be immediate benefits for the PDRA in being able to travel to the partner labs to carry out research, thus experiencing new working practices and acquiring new skills. This three-way collaboration will also seed future plans to attract European science funding to the UK.

Finally, the general public will benefit from the proposed research. Understanding the CRISPR/Cas enzymes will add to the general knowledge base of nucleic acid enzymes that informs many biomedical projects that, in turn, bring about improvements in human health. More directly, the design of new and safer synthetic enzymes for genome surgery will in the longer term contribute to economic and health quality of life of those living in the UK. We will also strive to increase the public's understanding of the science surrounding this project and of synthetic biology in general.

In addition to the traditional routes for publicising our results (peer-reviewed publications, web-based articles, seminars and conferences), we will be assisted in exploiting the knowledge and intellectual property generated by this project by the University of Bristol's Research and Development (RED) office. RED has multiple contacts with biotechnology and pharmaceutical companies. The development of collaborative links and the dissemination of results will also be assisted by the plans for Bristol University to be part of an Innovation and Knowledge Centre in Synthetic Biology; Mark Szczelkun and Mark Dillingham will both be actively involved in the centre. Bristol University and the School of Biochemistry are committed to public engagement activities, and we will continue to capitalise on these. Szczelkun has already built a hands-on mechanical model that can help explain the magnetic tweezers assay during out-reach activities.