Design of Circularly Permuted LOV2 Photoswitch for Optogenetic Control of Cellular Physiology and Cell-Based Therapy

Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly engineeredinto cell signaling pathways to remotely control protein activity and a myriad of physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we describe the engineering of a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. cpLOV2 can be used as a caging tag or an allosteric switch for insertion into host proteins. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate calcium channels, antagonize CRISPR-Cas9-mediated genome engineering, manipulate protein-protein interactions, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our synthetic biology approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.