The Opera Phenix Microscope for High Content Screening Applications

Lead Research Organisation: The Francis Crick Institute
Department Name: Research

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Description We have used the Opera Phenix system to investigate the function of genes identified as important in metastasis of pancreatic cancer. I particular, one gene implicated in pancreatic cancer is found to be involved in modifying the location of other proteins within the cell to the cell mebrane.
Exploitation Route While this work is very early stage, it is possible that the gene identified could provide a target for the develiopment of new therapies for pancreatic cancer, which is a highly lethal malignancy.
Sectors Healthcare

 
Description Details of emerging economic and societal impact arising from the award that you are reporting on (including how it has evolved) Measuring the ability of patient serum to efficiently neutralize and prevent a virus from infecting cells is historically a labour intensive, low throughput and slow assay requiring manual handling. It is however, a very informative assay but few if any high throughput pipelines exist internationally. The HTS science technology platform in collaboration with the Worldwide Influenza Centre (WIC) at the Crick have created and are running, a high throughput live-virus antibody neutralisation assay to measure the ability of patient serum to prevent cell infection by SARS-CoV-2 across thousands of samples simultaneously. At the heart of this assay is the ability to image and produce quantitative data from hundreds of 384-well plates, something which is only made possible by the Opera Phenix microscope. This unique neutralisation assay platform is essential to many ongoing clinical research studies such as -Legacy study (Crick/UCL) -VirusWatch/Thriva -SAFER (UCL/HSL) -NAOMI study on UK haemodialysis patients -SIREN study (PHE/UKHSA re-infection study) -CAPTURE study (Adaptive immunity and neutralising antibody activity against SARS-CoV-2 VOCs following COVID-19 vaccination in cancer patients) -WHO replacement IS and panel testing for VOCs This platform has the capacity to assay 1000 serum samples a week against three variants of concern. It is currently mapping the decline of neutralising antibody response in patients representing different vaccination regimes and should a new SARS-CoV-2 variant arise, it would be quickly possible to deploy the platform to estimate how vaccination history and previous infection might affect the ability of individuals to neutralise the new VOC with all of the implications for healthcare systems that such data would yield. A summary of how the findings from your award are impacting the public, private or third/voluntary sectors, and elsewhere This assay performed at this scale generates data of international significance that impacts public health decisions around the world and has led to a number of significant publications. Data generated on this platform are reported directly to national bodies such as UKSHA, JCVI and NERVTAG and has had a direct influence on vaccination policy for clinically important cohorts e.g. haemodialysis patients Challenges overcome to achieve impact Changing Covid priorities among funding bodies Significant impact within academia, for example the nucleation of a new research area Aside from 12 significant publications of direct impact on public health policy, the operation of this platform has led to the development of a separate Crick Covid Surveillance unit to continue to deliver and develop this platform, identifying and characterizing new variants before they become pandemic (in a manner analogous to the system in place for Influenza) References Basu S et al. 2021. Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase. The Biochemical journal 478: 2481-2497. Bertolin et al. 2021. Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase. Biochemical Journal 478: 2425-2443. Canal et al. 2021. Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease. Biochemical Journal 478: 2465- 2479. Carr et al. 2021. Neutralising antibodies after COVID-19 vaccination in UK haemodialysis patients. The Lancet. Carr et al. 2022 Neutralising antibodies against the Omicron variant after COVID-19 vaccination in UK haemodialysis patients. The Lancet Faulkner et al. 2021. Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains. Elife 10. Fendler et al. Functional antibody and T cell immunity following SARS-CoV-2 infection, including by variants of concern, in patients with cancer: the CAPTURE study. Nature Cancer. 2021a. Fendler et al. Adaptive immunity and neutralizing antibodies against SARS-CoV-2 variants of concern following vaccination in patients with cancer: the CAPTURE study. Nature Cancer. 2021b. Lim et al. 2021. Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease. Biochemical Journal 478: 2517- 2531. Lulla et al. Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome. J Virol. 2021;95(14):e0066321 Milligan et al. 2021. Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease. Biochemical Journal 478: 2499-2515. Wall et al. 2021a. AZD1222-induced neutralising antibody activity against SARS-CoV-2 Delta VOC. The Lancet. Wall et al. 2021b. Neutralising antibody activity against SARS-CoV-2 VOCs B.1.617.2 and B.1.351 by BNT162b2 vaccination. The Lancet. Wu et al. 2022. Three-dose vaccination elicits neutralising antibodies against SARS-CoV-2 Omicron variant. The Lancet. Fendler et al. 2022. Neutralising antibodies against SARS-CoV-2 Omicron VOC are undetectable after third vaccination in a substantial proportion of patients with blood cancer: The CAPTURE Study. The Lancet.
First Year Of Impact 2020
Sector Healthcare
Impact Types Societal,Policy & public services

 
Description COVID testing
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a national consultation
Impact Measuring the ability of patient serum to efficiently neutralize and prevent a virus from infecting cells is historically a labour intensive, low throughput and slow assay requiring manual handling. It is however, a very informative assay but few if any high throughput pipelines exist internationally. The HTS science technology platform in collaboration with the Worldwide Influenza Centre (WIC) at the Crick have created and are running, a high throughput live-virus antibody neutralisation assay to measure the ability of patient serum to prevent cell infection by SARS-CoV-2 across thousands of samples simultaneously. At the heart of this assay is the ability to image and produce quantitative data from hundreds of 384-well plates, something which is only made possible by the Opera Phenix microscope. This unique neutralisation assay platform is essential to many ongoing clinical research studies such as -Legacy study (Crick/UCL) -VirusWatch/Thriva -SAFER (UCL/HSL) -NAOMI study on UK haemodialysis patients -SIREN study (PHE/UKHSA re-infection study) -CAPTURE study (Adaptive immunity and neutralising antibody activity against SARS-CoV-2 VOCs following COVID-19 vaccination in cancer patients) -WHO replacement IS and panel testing for VOCs