Blue-shifted ancyromonad channelrhodopsins for multiplex optogenetics

Reviewer #1 (Public review):

Summary:

This work by Govorunova et al. identified three naturally blue-shifted channelrhodopsins (ChRs) from ancyromonads, namely AnsACR, FtACR, and NlCCR. The phylogenetic analysis places the ancyromonad ChRs in a distinct branch, highlighting their unique evolutionary origin and potential for novel applications in optogenetics. Further characterization revealed the spectral sensitivity, ionic selectivity, and kinetics of the newly discovered AnsACR, FtACR, and NlCCR. This study also offers valuable insights into the molecular mechanism underlying the function of these ChRs, including the roles of specific residues in the retinal-binding pocket. Finally, this study validated the functionality of these ChRs in both mouse brain slices (for AnsACR and FtACR) and in vivo in Caenorhabditis elegans (for AnsACR), demonstrating the versatility of these tools across different experimental systems.

In summary, this work provides a potentially valuable addition to the optogenetic toolkit by identifying and characterizing novel blue-shifted ChRs with unique properties.

Strengths:

This study provides a thorough characterization of the biophysical properties of the ChRs and demonstrates the versatility of these tools in different ex vivo and in vivo experimental systems. The mutagenesis experiments also revealed the roles of key residues in the photoactive site that can affect the spectral and kinetic properties of the channel.

Weaknesses:

While the novel ChRs identified in this work are spectrally blue-shifted, there still seems to be some spectral overlap with other optogenetic tools. The authors should provide more evidence to support the claim that they can be used for multiplex optogenetics and help potential end-users assess if they can be used together with other commonly applied ChRs. Additionally, further engineering or combination with other tools may be required to achieve truly orthogonal control in multiplexed experiments.

In the C. elegans experiments, partial recovery of pharyngeal pumping was observed after prolonged illumination, indicating potential adaptation. This suggests that the effectiveness of these ChRs may be limited by cellular adaptation mechanisms, which could be a drawback in long-term experiments. A thorough discussion of this challenge in the application of optogenetics tools would prove very valuable to the readership.

Reviewer #2 (Public review):

Summary:

Govorunova et al present three new anion opsins that have potential applications in silencing neurons. They identify new opsins by scanning numerous databases for sequence homology to known opsins, focusing on anion opsins. The three opsins identified are uncommonly fast, potent, and are able to silence neuronal activity. The authors characterize numerous parameters of the opsins.

Strengths:

This paper follows the tradition of the Spudich lab, presenting and rigorously characterizing potentially valuable opsins. Furthermore, they explore several mutations of the identified opsin that may make these opsins even more useful for the broader community. The opsins AnsACR and FtACR are particularly notable, having extraordinarily fast onset kinetics that could have utility in many domains. Furthermore, the authors show that AnsACR is usable in multiphoton experiments having a peak photocurrent in a commonly used wavelength. Overall, the author's detailed measurements and characterization make for an important resource, both presenting new opsins that may be important for future experiments, and providing characterizations to expand our understanding of opsin biophysics in general.

Weaknesses:

First, while the authors frequently reference GtACR1, a well-used anion opsin, there is no side-by-side data comparing these new opsins to the existing state-of-the-art. Such comparisons are very useful to adopt new opsins.

Next, multiphoton optogenetics is a promising emerging field in neuroscience, and I appreciate that the authors began to evaluate this approach with these opsins. However, a few additional comparisons are needed to establish the user viability of this approach, principally the photocurrent evoked using the 2p process, for given power densities. Comparison across the presented opsins and GtACR1 would allow readers to asses if these opsins are meaningfully activated by 2P.

Reviewer #3 (Public review):

Summary:

The authors aimed to develop Channelrhodopsins (ChRs), light-gated ion channels, with high potency and blue action spectra for use in multicolor (multiplex) optogenetics applications. To achieve this, they performed a bioinformatics analysis to identify ChR homologues in several protist species, focusing on ChRs from ancyromonads, which exhibited the highest photocurrents and the most blue-shifted action spectra among the tested candidates. Within the ancyromonad clade, the authors identified two new anion-conducting ChRs and one cation-conducting ChR. These were characterized in detail using a combination of manual and automated patch-clamp electrophysiology, absorption spectroscopy, and flash photolysis. The authors also explored sequence features that may explain the blue-shifted action spectra and differences in ion selectivity among closely related ChRs.

Strengths:

A key strength of this study is the high-quality experimental data, which were obtained using well-established techniques such as manual patch-clamp and absorption spectroscopy, complemented by modern automated patch-clamp approaches. These data convincingly support most of the claims. The newly characterized ChRs expand the optogenetics toolkit and will be of significant interest to researchers working with microbial rhodopsins, those developing new optogenetic tools, as well as neuro- and cardioscientists employing optogenetic methods.

Weaknesses:

This study does not exhibit major methodological weaknesses. The primary limitation of the study is that it includes only a limited number of comparisons to known ChRs, which makes it difficult to assess whether these newly discovered tools offer significant advantages over currently available options. Additionally, although the study aims to present ChRs suitable for multiplex optogenetics, the new ChRs were not tested in combination with other tools. A key requirement for multiplexed applications is not just spectral separation of the blue-shifted ChR from the red-shifted tool of interest but also sufficient sensitivity and potency under low blue-light conditions to avoid cross-activation of the respective red-shifted tool. Future work directly comparing these new ChRs with existing tools in optogenetic applications and further evaluating their multiplexing potential would help clarify their impact.