Temporal manipulation of genetic circuits in single cells

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Bio-imaging is a major tool in modern cell biology. A key feature has been the development of live cell imaging for the tracking and measurement of cellular processes such as cell division, cell death and the mechanisms by which signals are transmitted from the outside of the cell to control cellular behaviour. One major technology that has underpinned these developments has been the use of natural light emitting proteins: luciferases and fluorescent proteins. In order to understand complex cell systems it is also important to have specific manipulation technologies that can perturb key processes in clearly defined ways. Current interference technologies involve mutating or blocking gene activity, however these are not controllable or reversible. A major new technology has emerged from bacteria called CRISPR. It is a natural bacterial defence system that edits genes. The core of this system is a protein, Cas9, which can efficiently edit genes when targeted by guide RNAs that direct its activity to specific genes, and can incorporate and direct specific DNA sequence changes. Recently, new mutated versions of Cas9 have been developed that repress or activate genes without changing their sequence. We will now use a version of the Cas9 protein that has been split into two pieces. This can be fused to light sensitive proteins that bind together when you shine light on them. As a result, functional Cas9 is formed only when the cell is illuminated. Our aim is to use this approach to develop new ways to edit, repress and activate genes in response to illumination. This gives precisely timed control of gene perturbation, something that has previously been lacking. It will allow more precise investigation of gene function, a critical issue in modern biology. These tools will therefore be of great use in bio-imaging experiments and can have broader applications in biology and medicine.