The opsin engineering platform will combine genome mining and protein engineering with automated plasma membrane expression screenings and automated photocurrent measurements to develop optogenetic actuators (channelrhodopsins, ChRs) for optogenetic therapies. Moreover, the extensive dataset collected under standardized conditions will prospectively enable the most comprehensive elucidation of the channelrhodopsin fitness landscape, thus serving as a basis for data-driven protein engineering approaches that, in conjunction with structure-based approaches, will drive the development of the next generation of optogenetic actuators.

Figure: Opsin Engineering Platform (P1). The Opsin Engineering Platform (P1) will work closely with all Light2Treet platforms and teams to develop channelrhodopsins that combine a favorable risk profile with maximum efficiency. To this end, we will develop plasma membrane-localized, hypoimmunogenic channelrhodopsins with a red-shifted action spectrum and suitable kinetics. (A-B) Development of the next generation of optogenetic actuators using genome mining and protein engineering. (A) Identification of potentially suitable channelrhodopsins using phylogenetic analysis. (B) Structure-based protein engineering using high-resolution structures (ChR2 structure, PDB ID: 6EID), 3D models (AlphaFold, DeepMind), and cryo-EM analyses (Titan Krios G4, University of Göttingen cryo-EM platform). (C-D) The application of automated methods enables the investigation of a multitude of natural and mutagenesis-optimized channelrhodopsins. The channelrhodopsin analysis pipeline is based on (C) an automated spinning disk confocal microscope (CQ1, Yokogawa) for channelrhodopsin plasma membrane expression screenings and a patch-clamp robot (Opto-SyncroPatch 384, Nanion) for automated photocurrent measurements. (E) Exemplary ChReef photocurrent measurement on the Opto-SyncroPatch 384.