CRISPR-mediated chromosome-specific labelling for live cell imaging applications

Genomes carry the information to build and regulate organisms. They are elaborate physical structures comprising DNA that undergoes dramatic dynamic changes throughout the cell lifecycle. In humans, the DNA is packaged into 23 discrete structures called chromosomes. These have been studied for many decades in live cells using light and fluorescence microscopy. However, in these techniques all of the chromosomes are indistinguishable from each other. This limitation has held back the study of many problems in chromosome biology. In this proposal, we aim to develop a range of cell lines suitable for live cell studies, each with a single known chromosome fluorescently labelled. To accomplish this, we will use a modification of the recently developed CRISPR method which allows the targeting of a DNA cutting enzyme called Cas9 to anywhere on the genome by introducing a specific piece of RNA. We will use a modified version of Cas9 where the cutting activity has been disabled such that it remains stuck to the chosen location on the genome. By attaching fluorescent proteins to the modified Cas9 and directing many copies to a specific location on a chosen chromosome we will construct a 'barcoding' system with unique chromosome identifiers compatible with state-of-the-art live cell imaging. This technology will open up new avenues of research into, for example, how chromosomes are arranged in the nucleus, how chromosomes are packaged and how they are segregated during cell division.

While we are able to investigate the dynamic behaviour of chromosomes as a collective or individually but without identification, studying specific chromosomes in live cells is not straightforward at the present time. The ability to easily confirm the identity of chromosomes in living cells will revolutionize various fields of study in chromosome and cancer biology such as nuclear organisation and chromosome territories, chromosome dynamics, chromosome instability and its effect on cell fate. Moreover, being able to visualise multiple genomic loci within a single chromosome will pioneer research in dynamic chromatin organisation during cellular processes like DNA replication, transcription and repair. We propose to develop a state-of-the-art assay using fluorescently labelled Cas9 orthologues and multiple guide RNAs (gRNAs) to uniquely label each human chromosome with multi-coloured barcodes. We will develop a stand-alone Python-based bioinformatics tool for automatically choosing the optimal tiling array of gRNAs within a given genomic region that will accommodate for the different requirements of Cas9 bacterial orthologues. Using high throughput and multiplex cloning we will produce gRNA expression cassettes and use those to produce lentiviral particles. We will then construct a battery of near-diploid RPE cell lines with single chromosomes labelled, with the possibility of extending this to multiple chromosomes per cell line. The cell lines will be verified using live cell imaging, FISH and whole genome sequencing.

The general public
The chromosome labelling tools developed in this project will enable novel studies in many areas of chromosome biology, including nuclear architecture, chromosome dynamics, chromosome missegregation and cell fate. The consequent advances in basic scientific knowledge will help support the advancement of medicine which benefits the general population both in terms of health, well-being, and indirectly on the socio-economic state of the United Kingdom.

Translational medicine
Results from basic research are converted into tangible patient benefits in the field of translational medicine. Although this research proposal is not directly linked to translational medicine, areas of research that stand to directly benefit, such as chromosome missegregation and chromosome instability are crucially linked to tumour evolution in cancer. Therefore, the resources we develop in this project will provide benefits to both basic and translation science and will generate knowledge that has the potential to lead to new translational programmes in the future.

Science outreach
Public science outreach activities are vital for making the general public aware of scientific advances and understanding how science progresses. As laboratories, we intend to fully participate in the outreach activities run by UCL Cancer Institute, which involve school visits, demonstrating microscopy, discussing science with students, and other activities to engage with the wider community. The labelled chromosomes developed in this project will offer a fascinating demonstration of advanced microscopy techniques for outreach events.

Academic impact
The approach taken in this project will also have an impact in the academic environment by providing further insight into the use of CRISPR/Cas9 methods in cell biology. The availability of the bank of chromosome labelled cell lines and the associated plasmids for generating similar resources in other cell types will have a significant impact on researchers studying chromosome behaviour.

The project will employ a Technician, who will gain additional or enhanced skills in cell culture, molecular biology and microscopy, in addition to academic writing and presentation skills. After completion, the Technician will be able to use their skills to contribute to academia, in the host lab or elsewhere, or to the pharmaceutical or biotechnology industries.