The Opera Phenix Microscope for High Content Screening Applications

The Crick is a partnership between the Medical Research Council, Cancer Research UK, the Wellcome Trust and three leading universities: UCL (University College London), Imperial College London and King's College London. The Crick aspires to be one of the world's leading medical research institutes. The Crick achieves operational and research efficiencies and economies-of-scale through centralised facilities and functions, known as Science Operations, that provide all researchers at the Crick, irrespective of affiliation, with access to cutting-edge equipment, animals for research and laboratory enabling functions such as the High Throughput Screening facility (HTS).

Technical advances mean that it is now commonplace to be able to identify when genes have been altered or lost as part of ageing or a disease process. It is less obvious what effect this loss has on any individual cell and how it contributes to a disease. Indeed, understanding the functional roles for any gene in any particular cell or condition or how any gene normally contributes to the way a cell behaves or responds is still somewhat lacking. Fortunately, we now have reagents (called CRISPRs) that allow researchers to inactivate any gene of interest and assess the consequences of its loss on cell behaviour. The effect of gene loss on the cell is inferred from quantitative analysis of images taken of the cells when they are either growing and moving normally or when they have been challenged to respond to a stimulus. By measuring the properties of cells lacking a single gene, researchers can infer something about the role it might be playing in the cell as a whole. The HTS facility at the Crick has created a system that allows researchers to study the effect of gene loss on cell behaviour for hundreds to thousands of genes simultaneously, experiments referred to as screening.

This proposal is for a microscope capable of automatically imaging and analysing the tens of thousands of samples that such experiments demand. This new microscope in conjunction with the CRISPR reagents gives us the opportunity to shed light on both the basic mechanisms of how genes control what cells do and potentially the role they play in important human diseases.

The Crick aspires to be one of the world's leading medical research institutes. It achieves research efficiencies through centralised facilities and functions such as the High Throughput Screening Scientific Technology Platform (STP). A BBSRC-funded high content imaging microscope would be placed in the High Throughput Screening STP and made available to 100+ research groups at the Crick, and their collaborators. This application is being made by several Crick Group Leaders (Downward, Swanton, Boulton, Frickel, Tooze and Ciccarelli). These diverse projects (illustrative of the breadth of research at the Crick), are united by the need for high content imaging to fully exploit advances in gene engineering.

Downward- Quantitative imaging of sub-cellular localisation of tagged Ras family members in response to CRISPR knockout of palmitoyl acyltransferases.

Swanton- Creating an automated 'smart' confocal acquisition platform to identify and classify chromosomal abberations and mitotic errors arising in response to CRISPR gene knockout.

Boulton- Using arrayed CRISPR screening and confocal imaging of PML bodies co-localising to telomeres to Identify genes required for alternative lengthening of telomeres.

Frickel-Using arrayed CRISPR screening in iPS-derived macrophages and quantitative confocal imaging to identify ubiquitin modification enzymes combating Toxoplasma in human macrophages.

Tooze- Creation of reporter cell lines to measure the dynamic interactions underpinning the initiation of autophagy and using arrayed CRISPR screening and live cell confocal imaging of these lines to identify new regulatory proteins of the process.

Ciccarelli-Creation of a pipeline of confocal acquisition and multi-parametric analysis for the phenotypic characterisation of CRISPR knockout cell lines.

The Crick aspires to be one of the world's leading medical research institutes. Its multidisciplinary approach, an emphasis on practical application of research and its links with academia, industry and the public sector will speed up the translation of discoveries made in the laboratory into treatments for disease. We fully expect outcomes from the Opera Phenix microscope system and its use by researchers across the Crick to have a pathway to impact through academic, economic and societal impacts.

Academic impact:
- Publication via various media including papers and conference presentations;
- Knowledge transfer and education, especially with respect to PhD students and early-career researchers / post-doctoral research associates.

Economic and societal impact:
- The Crick, its founding partners (the Medical Research Council, Cancer Research UK, the Wellcome Trust, UCL (University College London), Imperial College London and King's College London), and legacy research institutes at Mill Hill (MRC), Lincoln's Inn Fields and Clare Hall (CRUK) all have strong track records for converting research and discoveries into economic and societal benefit;
- The Crick organisation includes well-funded and specific groups responsible for Translation, Clinical Research, Public Engagement and Education, thus we are confident that research outcomes from the BBSRC funded Opera Phenix microscope will convert into economic and societal benefits.

It is worth emphasising that the BBSRC funded Opera Phenix microscope system will be housed in the Crick's High Throughput Screening STP and hence available to more than 100 research groups such that the pathways to impact are many and varied.