Synthetic strategies for optically controlling gene expression may enable the precise spatiotemporal control of genes in any combination of cells that cannot be targeted with specific promoters. We develop an improved genetic code expansion system in Caenorhabditis elegans and use it to create a photoactivatable Cre recombinase. We laser-activate Cre in single neurons within a bilaterally symmetric pair to selectively switch on expression of a loxP-controlled optogenetic channel in the targeted neuron. We use the system to dissect, in freely moving animals, the individual contributions of the mechanosensory neurons PLML/PLMR to the C. elegans touch response circuit, revealing distinct and synergistic roles for these neurons. We thus demonstrate how genetic code expansion and optical targeting can be combined to break the symmetry of neuron pairs and dissect behavioural outputs of individual neurons that cannot be genetically targeted.
Keywords: C. elegans; Cre recombinase; biochemistry; chemical biology; genetic code expansion; neuroscience; non canonical amino acids; optogenetics; photo-caged amino acid.
Animal behaviour and movement emerges from the stimulation of nerve cells that are connected together like a circuit. Researchers use various tools to investigate these neural networks in model organisms such as roundworms, fruit flies and zebrafish. The trick is to activate some nerve cells, but not others, so as to isolate their specific role within the neural circuit. One way to do this is to switch genes on or off in individual cells as a way to control their neuronal activity. This can be achieved by building a photocaged version of the enzyme Cre recombinase which is designed to target specific genes. The modified Cre recombinase contains an amino acid (the building blocks of proteins) that inactivates the enzyme. When the cell is illuminated with UV light, a part of the amino acid gets removed allowing Cre recombinase to turn on its target gene. However, cells do not naturally produce these photocaged amino acids. To overcome this, researchers can use a technology called genetic code expansion which provides cells with the tools they need to build proteins containing these synthetic amino acids. Although this technique has been used in live animals, its application has been limited due to the small amount of proteins it produces. Davis et al. therefore set out to improve the efficiency of genetic code expansion so that it can be used to study single nerve cells in freely moving roundworms. In the new system, named LaserTAC, individual cells are targeted with UV light that ‘uncages’ the Cre recombinase enzyme so it can switch on a gene for a protein that controls neuronal activity. Davis et al. used this approach to stimulate a pair of neurons sensitive to touch to see how this impacted the roundworm’s behaviour. This revealed that individual neurons within this pair contribute to the touch response in different ways. However, input from both neurons is required to produce a robust reaction. These findings show that the LaserTAC system can be used to manipulate gene activity in single cells, such as neurons, using light. It allows researchers to precisely control in which cells and when a given gene is switched on or off. Also, with the improved efficiency of the genetic code expansion, this technology could be used to modify proteins other than Cre recombinase and be applied to other artificial amino acids that have been developed in recent years.
© 2021, Davis et al.