Temporal manipulation of genetic circuits in single cells

Bio-imaging is a major tool in modern cell biology. A key feature has been the development of live cell imaging for the tracking and measurement of cellular processes such as cell division, cell death and the mechanisms by which signals are transmitted from the outside of the cell to control cellular behaviour. One major technology that has underpinned these developments has been the use of natural light emitting proteins: luciferases and fluorescent proteins. In order to understand complex cell systems it is also important to have specific manipulation technologies that can perturb key processes in clearly defined ways. Current interference technologies involve mutating or blocking gene activity, however these are not controllable or reversible. A major new technology has emerged from bacteria called CRISPR. It is a natural bacterial defence system that edits genes. The core of this system is a protein, Cas9, which can efficiently edit genes when targeted by guide RNAs that direct its activity to specific genes, and can incorporate and direct specific DNA sequence changes. Recently, new mutated versions of Cas9 have been developed that repress or activate genes without changing their sequence. We will now use a version of the Cas9 protein that has been split into two pieces. This can be fused to light sensitive proteins that bind together when you shine light on them. As a result, functional Cas9 is formed only when the cell is illuminated. Our aim is to use this approach to develop new ways to edit, repress and activate genes in response to illumination. This gives precisely timed control of gene perturbation, something that has previously been lacking. It will allow more precise investigation of gene function, a critical issue in modern biology. These tools will therefore be of great use in bio-imaging experiments and can have broader applications in biology and medicine.

The CRISPR technology has had a major impact on the field of targeted gene editing, and is now starting to provide new approaches for targeted modulation of gene expression. At the same time optogenetics became a major field in neuroscience, but the approaches clearly have broader potential impact across cell biology. In bio-imaging the preparation of precisely and non-invasively labelled samples is very important, and there is also a need for better manipulation technologies that can be temporally controlled. Here, we propose to further develop emerging approaches for the use of photo-activatable control of the Cas9 protein, which is the central editing enzyme in the CRISPR process. Specifically we will develop improved expression cassettes for wild-type Cas9 as well as dead Cas9 (mutated to kill the editing activity). Dead Cas9 will also be fused to the KRAB chromatin-silencing domain and the VPR transcriptional activation domain. The utility of these tools will be carefully assessed, including the use of fluorescence cross-correlation spectroscopy to measure the efficiency of re-association of the split Cas9 in response to photo-activation. The efficacy of these new split Cas9 proteins will be tested using guide RNAs directed to the luciferase reporter gene, which has a short half-life and is easily quantifiable. In particular, we will use cells expressing a TNFalpha reporter cassette, since we have both cell lines and transgenic animals available for studies of expression of this important inflammatory cytokine. In addition the technology will also be tested for manipulation of the key IkappaBalpha and A20 feedback loops, to further test their roles in the control of NF-kappaB oscillations. Finally, we have identified a series of other users who work on a variety of different systems, who have expressed an interest in early access to this technology.

This project will develop a defined set of technologies of use to many biomedical scientists in academia and industry. Mike White is the recently appointed Domain Lead for Platform Sciences and Technologies in the new Faculty of Biology, Medicine and Health in Manchester. In this role he is a champion for technology in the faculty and coordinates networks of technology developers to ensure added value. Through his role as Director of the Systems Microscopy Centre, Mike White already leads training courses in bio-imaging which are supported by Zeiss. Antony Adamson is the local expert in CRISPR, and plays a major role in encouraging and enhancing the use of this technology across the faculty. He provides training talks and sends out regular newsletters. We therefore are very well placed to encourage local use of the technology. A group of users have provided supportive letters indicating the potential application to a set of important biological systems. As well as local communication of the technology we will aim to publish the results, share the constructs and present the results at national and international meetings. We have already identified a set of likely user groups and meetings where this work could be presented. These include the NF-kappaB community, as this will be the first system on which the technology will be tested. We also have excellent contacts with industry; Zeiss and Hamamatsu have been long-term collaborators with Mike White, and currently provide substantial funds for our training and outreach activities. In addition, we have a longstanding tie with AstraZeneca (and a new recent project to study cardiomyocyte toxicity). Mike White also supervisors a GSK funded studentship. Through the Manchester Collaborative Centre for Inflammation Research there are general ties and collaborative agreements with AstraZeneca and GSK. Further to this, bio-imaging is very visual and therefore offers considerable potential for effective public dissemination. We will present data at an open day, local exhibitions and in schools. We hope to arrange an undergraduate communication project to aid the development of these tools. Finally, we will plan an application to the Royal Society Summer Exhibition (application Oct 2017). General outreach is co-ordinated through our assistant Penny Titterington. Overall this project offers great potential to develop an achievable step change in technology that we are well placed to exploit. There is potential for this to have a significant impact in the bio-imaging and systems biology communities.