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

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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.