Next Generation Platform for Single Cell Genetic Manipulation and Analysis

In the last decade, scientists have established new ways to study and understand human biology and the basis of disease. CRISPR-Cas gene editing is a technology which can be used to change the activity of genes and gene networks, which allows researchers to measure biological processes more accurately. With this technology we can 'knock out' genes to explore then function once they have been removed. Or we can use gene editing o tag genes with useful functional proteins, such as fluorescent proteins from Jellyfish, allowing us to watch what genes and proteins do in real time. Gene editing is being performed in lots of different cell models, from cancer cell lines, to induced Pluripotent Stem Cells (iPSCs) and human Embryonic Stem Cells (hESC) which can be treated with drugs to turn them into different tissues in a dish known as organoid cultures. We can also gene edit primary cells like neurons and immune cells, which have been used directly from human patients. However there are still challenges preventing the successful application of CRISPR-Cas9 in many of these cell types. For example, it can be difficult, and expensive, to deliver gene editing reagents to stem cells and primary cells, as well as isolating single cells afterwards. Some cell types, like immune cells, need to be activated or expanded before they can be manipulated which can affect the outcome of the experiment. Even with standard cultured cell lines, the process is labour intensive and costly.

The FluidFM OMNIUM is a technology developed to help with the bottlenecks and limitations often encountered in cell based research. It is a device that is capable of injecting delivering materials, such as CRISPR reagents directly into the nucleus of single cells, which improves the efficiency of gene editing in a variety of cell types, including stem cells and primary cells. It also allows for more effective isolation of single cells with gentle handling. Together, this makes it possible to create clonal cultures of gene edited cells more quickly and efficiently, using significantly less reagents and other resources, and requiring less time and effort from researchers.

Further, advances in single-cell sequencing and imaging technologies have allowed researchers to study individual cells to learn more about immunology, neuroscience, cancer, and development. But current methods of single-cell profiling, such as measuring the level of RNA, the building block of proteins in a cell, requires cell lysis, which means that repeated analysis on the same cell cannot be done. This means we cannot measure what changes over time in a cell response to a stimulation. The FluidFM OMNIUM platform not only allows for injection of reagents into cells but it also allows for the extraction of a small amount of material from live cells. With this ability, it is possible to monitor how cells respond over several time scales to various perturbations, something which was not achievable before.

These technologies will have use across a range of study areas of biology and disease, in particular genetic disease, immunology, cancer and development.

We request funds to purchase a FluidFM Omnium, to be housed in the Genome Editing Unit (GEU) core facility at the University of Manchester (UoM). This will be used for genetic manipulation of cell models, including cell lines, primary cells and stem cells. It will also be used for sampling of cellular materials for downstream single cell transcriptomics. The equipment will be made available for internal and external research groups working in MRC strategic priority areas, and will be operated by specialist Research Technical Professionals, with a cost-recovery operating model to ensure long-term accessibility and maintenance.
The FluidFM is a state of the art system that operates based on force-controlled cantilevers that can directly inject nuclei of single-cells with CRISPR-Cas9 complexes for high efficiency gene editing in single cells, and dramatically reducing reagent costs and researcher labour. This is a step change in how we make genetically modified cell lines and will greatly increase our capacity and throughput. The system is capable of genetic manipulation in difficult to transfect cell types such as primary cells, and also stem cells, expanding the breadth of service available from the GEU.
The FluidFM can also extract cytoplasmic material from live cells for downstream analysis. Picolitres can be repeatedly extracted from cells or from targeted cellular regions without compromising cell viability. The extracted biopsy can then be deposited into droplets within the platform and then processed for transcriptomic analysis externally in a technique termed Live-Seq.
This multi-functional, cutting edge system will dramatically expand the range of experiments available to researchers, and also the cell types and models that can be used. Because of its high precision and versatility, the FluidFM has the potential to enable new research and applications in a wide range of fields including nanotechnology, materials science and microfluidics.