EXPANDING THE OPTOGENETIC TOOLBOX: GENOME MINING AND AUTOMATED CHARACTERIZATION BASED DISCOVERY OF RED-SHIFTED CHANNELRHODOPSINS

Optogenetics has profoundly impacted neuroscience by enabling precise, light-mediated control of neuronal circuits with high spatiotemporal resolution. Central to this technology are Channelrhodopsins (ChRs), light-gated ion channels. Over the past two decades, numerous ChR variants have been discovered and optimized. However, optogenetic stimulation in vivo remains challenging, particularly for applications requiring high-frequency activation, such as the auditory system. A key priority in addressing these challenges is the discovery of red-shifted ChRs, which reduce the risk of phototoxicity and allow dual-colour experiments in combination with blue-light-activated tools. In this study, we screened 50 previously uncharacterized ChRs from green algae, targeting sequences with amino acid residues linked to red-shifting. Using automated photocurrent measurements and high-throughput spinning disk confocal microscopy, we focused on residues influencing the polarity at the β-ionone ring, the retinal-binding pocket geometry, and the counterion complex. Our screening identified four promising red-shifted candidates, including what is, to our knowledge, the most red-shifted depolarizing natural ChR discovered to date with peak sensitivity at 610 nm. Toward achieving ultrafast closing kinetics, we identified a green-light-activated ChR with a τ_off of 2.5 ± 0.3 ms, which likely represents the fastest natural variant observed yet. These findings significantly expand the optogenetic toolbox, offering new possibilities for high-frequency neural coding and multi-wavelength circuit interrogation. By providing improved tools for both speed and spectral range, this work contributes to advancing the potential for optogenetic applications in both fundamental neuroscience and future clinical interventions, such as sensory restoration.