Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via a mechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Escherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29°C but negligible activity at 37°C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity.

All synapses require fusion-competent vesicles and coordinated Ca2+-secretion coupling for neurotransmission, yet functional and anatomical properties are diverse across different synapse types. We show that the presynaptic protein RIM-BP2 has diversified functions in neurotransmitter release at different central murine synapses and thus contributes to synaptic diversity. At hippocampal pyramidal CA3-CA1 synapses, RIM-BP2 loss has a mild effect on neurotransmitter release, by only regulating Ca2+-secretion coupling. However, at hippocampal mossy fiber synapses, RIM-BP2 has a substantial impact on neurotransmitter release by promoting vesicle docking/priming and vesicular release probability via stabilization of Munc13-1 at the active zone. We suggest that differences in the active zone organization may dictate the role a protein plays in synaptic transmission and that differences in active zone architecture is a major determinant factor in the functional diversity of synapses.

1All synapses require fusion--competent vesicles and coordinated Ca 2+ --secretion coupling for 2 neurotransmission, yet functional and anatomical properties show a high diversity across different 3 synapse types. We show here that the presynaptic protein RIM--BP2 has diversified functions in 4 neurotransmitter release at different central mammalian synapses and thus contributes to 5 synaptic diversity. At hippocampal pyramidal CA3--CA1 synapses, RIM--BP2 loss has a mild effect on 6 neurotransmitter release, by only regulating Ca 2+ --secretion coupling. However, at hippocampal 7 mossy fiber synapses RIM--BP2 has a strong impact on neurotransmitter release by promoting 8 vesicle docking/priming via recruitment of Munc13--1. In wild type mossy fiber synapses, the 9 distance between RIM--BP2 clusters and Munc13--1 clusters is larger than in hippocampal pyramidal 10 CA3--CA1 synapses, suggesting that spatial organization may dictate the role a protein plays in 11 synaptic transmission and that differences in active zone architecture is a major determinant factor 12 in the functional diversity of synapses . 13 14 15 16 17 18

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