Abstract
BACKGROUND: Our ability to engineer opsins is limited by an incomplete understanding of how sequence variations influence function. The vastness of opsin sequence space makes systematic exploration difficult. NEW METHOD: In recognition of the need for datasets linking opsin genetic sequence to function, we pursued a novel method for screening channel-rhodopsins to obtain these datasets. In this method, we integrate advances in robotic intracellular electrophysiology (Patch) to measure optogenetic properties (Excite), harvest individual cells of interest (Pick) and subsequently sequence them (Sequence), thus tying sequence to function. RESULTS: We used this method to sequence more than 50 cells with associated functional characterization. We further demonstrate the utility of this method with experiments on heterogeneous populations of known opsins and single point mutations of a known opsin. Of these point mutations, we found C160W ablates ChrimsonR's response to light. CONCLUSION AND COMPARISON TO EXISTING METHODS: Compared to traditional manual patch clamp screening, which is labor-intensive and low-throughput, this approach enables more efficient, standardized, and scalable characterization of large opsin libraries. This method can enable opsin engineering with large datasets to increase our understanding of opsin sequence-function relationships.