High-performance microbial opsins for spatially and temporally precise perturbations of large neuronal networks

The biophysical properties of existing optogenetic tools constrain the scale, speed, and fidelity of precise optogenetic control. Here, we use structure-guided mutagenesis to engineer opsins that exhibit very high potency while retaining fast kinetics. These new opsins enable large-scale, temporally and spatially precise control of population neural activity. We extensively benchmark these new opsins against existing optogenetic tools and provide a detailed biophysical characterization of a diverse family of opsins under two-photon illumination. This establishes a resource for matching the optimal opsin to the goals and constraints of patterned optogenetics experiments. Finally, by combining these new opsins with optimized procedures for holographic photostimulation, we demonstrate the simultaneous coactivation of several hundred spatially defined neurons with a single hologram and nearly double that number by temporally interleaving holograms at fast rates. These newly engineered opsins substantially extend the capabilities of patterned illumination optogenetic paradigms for addressing neural circuits and behavior. [Display omitted] •ChroME2.0 opsins enable large-scale, temporally precise optogenetic control•ChroME2s allows large ensemble 2p stimulation of >600 neurons per second in vivo•Detailed biophysical analysis of the current opsin toolbox with 2p excitation Using structure-guided design, the authors develop second-generation ChroME-based cation channel rhodopsins that exhibit extremely high potency while preserving fast kinetics, thereby expanding the optogenetic toolbox. ChroME2.0 opsins permit spatially and temporally precise two-photon holographic neural control at unprecedented scales, a key technological step forward for understanding brain circuits and behavior.