Ultrapotent channelrhodopsin variants for optogenetic applications

Abstract

The present disclosure provides channelrhodopsin variants that are ultrapotent optogenetic actuators of cellular electrical activity. These variants show significantly increased photocurrent magnitude and light sensitivity compared to channelrhodopsin ChRmine (SEQ ID NO:1) from Rhodomonas lens.

Description

ULTRAPOTENT CHANNELRHODOPSIN VARIANTS FOR OPTOGENETIC APPLICATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 699,006, filed September 25, 2024, which application is incorporated herein by reference in its entirety.INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING XML

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 544WO_SEQ_LIST” created on September 17, 2025, and having a size of 60,346 bytes. The contents of the Sequence Listing X L are incorporated by reference herein in their entirety.INTRODUCTION

[0003] Optogenetic approaches enable control of cellular electrical activity with light by expression of light-sensitive proteins, including microbial light-gated ion channels (channelrhodopsins), in genetically targeted cells1 2. Channelrhodopsins are multimeric proteins with one conduction pathway per protein chain. Each subunit consists of seven transmembrane helices and a retinal chromophore covalently bound through a Schiff base to a conserved lysine residue. In the dark, retinal adopts an all-trans configuration and the ion channel is closed. Light stimulation results in retinal isomerization to the 13-cis configuration and corresponding protein conformational changes that open a pore for ion conduction across the cell membrane34. Channelrhodopsins vary in their functional properties including ion selectivity, kinetics, light sensitivity, spectral sensitivity, conductance, and expression level in cells. While many channelrhodopsins have been characterized, control of neural electrical signals with high spatial and temporal fidelity in a minimally invasive manner is still limited by intrinsic biophysical properties of reported channelrhodopsins.

[0004] There is a need for improved channelrhodopsins that are potent activators of cellular electrical activity.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with Government support under GM 148599, awarded by the National Institutes of Health. The Government has certain rights in the invention.SUMMARY

[0006] The present disclosure provides channelrhodopsin variants that are ultrapotent optogenetic actuators of cellular electrical activity. These variants show significantly increased photocurrent magnitude and light sensitivity compared to channelrhodopsin ChRmine (SEQ ID NO:1) from Rhodomonas lens.

[0007] In certain aspects, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a channelrhodopsin (ChRmine) variant comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:1 and comprising one or more of the following substitutions relative to SEQ ID NO:1 : i. an amino acid substitution of the alanine at amino acid 81 (A81); ii. an amino acid substitution of the leucine at position 47 (L47); iii. an amino acid substitution of the glutamine at position 130 (Q130); iv. an amino acid substitution of the threonine at position 119 (T119); and v. an amino acid substitution of the serine at position 35 (S35), wherein the amino acid positions are numbered based on the numbering of SEQ ID NO:1.

[0008] The ChRmine variants, of the present disclosure, when expressed in cells may be post-translationally modified such that the N-terminal methionine is removed and the alanine at position 2 is acetylated.

[0009] Additional aspects of the present disclosure include vectors encoding the disclosed variants and kits for expressing the disclosed variants and cells expressing the disclosed variants.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1. Cryo-EM structures of ChRmine (SEQ ID NO:1) in dark and light- stimulated states reveal targets for structure-based engineering of channel properties.

[0011] FIGS. 2A and 2B. Photocurrents recorded from new channelrhodopsin variants.

[0012] FIG. 3. Light sensitivity of new channelrhodopsin variants.

[0013] FIGS. 4A and 4B. Closing rate recorded from new channelrhodopsin variants.

[0014] FIGS. 5A and 5B. Closing rate recorded from new channelrhodopsin variants.

[0015] FIG. 6. Desensitization of channelrhodopsin variants.

[0016] FIG. 7. Recovery from inactivation of channelrhodopsin variants.DETAILED DESCRIPTION

[0017] The present disclosure provides channelrhodopsin variants that are ultrapotent optogenetic actuators of cellular electrical activity. These variants show significantly increased photocurrent magnitude and light sensitivity compared to channelrhodopsin ChRmine (SEQ ID NO:1) from Rhodomonas lens.

[0018] In certain aspects, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a channelrhodopsin variant comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:1 and comprising one or more of the following substitutions relative to SEQ ID NO:1 : i. an amino acid substitution of the alanine at amino acid 81 (A81); ii. an amino acid substitution of the leucine at position 47 (L47); ill. an amino acid substitution of the glutamine at position 130 (Q130); iv. an amino acid substitution of the threonine at position 119 (T119); and v. an amino acid substitution of the serine at position 35 (S35), wherein the amino acid positions are numbered based on the numbering of SEQ ID NO:1.

[0019] Additional aspects of the present disclosure include the ChRmine variants and vectors encoding the disclosed variants and kits for expressing the disclosed variants and cells expressing the disclosed variants.

[0020] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0021] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0022] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0024] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0025] It is noted that, as used herein and in the appended claims, the singular forms “a", “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0026] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0027] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.DEFINITIONS

[0028] “Derived from” in the context of an amino acid sequence or polynucleotide sequence is meant to indicate that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid, and is not meant to be limiting as to the source or method in which the protein or nucleic acid is made.

[0029] The terms "polypeptide", and "protein" are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acid residues are usually in the natural "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. In addition, the amino acids, in addition to the 20 "standard" amino acids, include modified and unusual amino acids, which include, but are not limited to those listed in 37 CFR (§1.822(b)(4)). Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates either a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group. However, the absence of a dash should not be taken to mean that such peptide bonds or covalent bond to a carboxyl or hydroxyl end group is not present, as it is conventional in representation of amino acid sequences to omit such. The term "peptide” also refers to a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues but is generally shorter than a protein or a polypeptide, e.g., less than 50 amino acids long, e.g., 2-50 amino acids in length. The terms protein, polypeptide, and peptide may be used interchangeably.

[0030] As used herein, the term "binding" refers to the non-covalent interactions of the type which occur between two molecules. The strength or affinity of binding interactions can be expressed in terms of the dissociation constant (KD) of the interaction, wherein a smaller KDrepresents a greater affinity. Binding properties of selected polypeptides can be quantified using methods well known in the art.

[0031] “Isolated” refers to an entity of interest that is in an environment different from that in which the entity may naturally occur or is initially produced in. An “isolated” compound (e.g., an “isolated” polypeptide) is separated from all or some of the components that accompany it and may be substantially enriched, e.g., may be purified so that the compound is at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99%, or greater than 99% pure, or free of impurities, contaminants, and / or components other than the compound. “Isolated” also refers to the state of a compound separated from all or some of the components that accompany it during manufacture (e.g., chemical synthesis, recombinant expression, culture medium, and the like).

[0032] As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[0033] In all embodiments of polypeptides disclosed herein, any N-terminal methionine residues are optional (i.e., the N-terminal methionine residue may be present or absent). In all embodiments, the ChRmine variants may include post-translational modification(s), e.g., acetylation (at e.g., Ala at position 2 with reference to SEQ ID NO:1), deletion of N-terminal amino acid and / or C-terminal amino acid, glycosylation, etc.

[0034] The term "conservative substitution" is used in reference to proteins to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) of the molecule. Typically, conservative amino acid substitutions involve substituting one amino acid for another amino acid with similar chemical properties (e.g., charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). The polypeptides encompassed by the present disclosure include those that have one or more conservative substitutions relative to the amino acid sequences provided here.

[0035] Percent identity between a pair of sequences may be calculated by multiplying the number of matches in the pair by 100 and dividing by the length of the aligned region,including gaps. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another. Only internal gaps are included in the length, not gaps at the sequence ends. Percent Identity = (Matches x 100) / Length of aligned region (with gaps).

[0036] Numeric ranges are inclusive of the numbers defining the range.

[0037] The terms "illuminating" or "illumination" used herein with respect to a cell or a tissue refer to exposing (or exposure of) said cell or said tissue to light. Illumination of a cell comprising a variant as disclosed herein in its plasma membrane typically results in the movement of cations through the plasma membrane of the cell in response to the light (i.e. , a photocurrent). This may result in the depolarization of a nerve cell membrane and triggering of action potentials.

[0038] As used herein, the term “vector,” can refer to a vehicle for carrying or transferring a nucleic acid. Non-limiting examples of vectors include viral vectors (for example, adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, lentiviral vectors, herpes virus vectors, phages, and poxvirus vectors); non-viral vectors such as liposomes, naked DNA, plasmids, cosmids; and the like.

[0039] As used herein, the term “variant” refers to a polynucleotide or polypeptide having a sequence substantially similar to a reference (e.g., the parent) polynucleotide or polypeptide. In the case of a polynucleotide, a variant can have deletions, substitutions, additions of one or more nucleotides at the 5' end, 3' end, and / or one or more internal sites in comparison to the reference polynucleotide. Similarities and / or differences in sequences between a variant and the reference polynucleotide can be detected using conventional techniques known in the art, for example polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis. Generally, a variant of a polynucleotide, including, but not limited to, a DNA, can have at least, or at least about, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference polynucleotide as determined by sequence alignment programs known in the art. In the case of a polypeptide, a variant can have deletions, substitutions, additions of one or more amino acids in comparison to the reference polypeptide. Similarities and / or differences in sequences between a variant and the reference polypeptide can be detected using conventional techniques known in the art, for example Western blot. A variant of a polypeptide can have, for example, at least, or at least about, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference polypeptide as determined by sequence alignment programs known in the art.

[0040] The term “naturally occurring” or “wild type” as used herein refers to materials which are found in nature or a form of the materials that is found in nature.

[0041] As used herein, a “subject” refers to an animal that is the object of treatment, observation or experiment. “Animal” includes cold- and warm-blooded vertebrates (e.g., mammals) and invertebrates (e.g., fish, shellfish and reptiles). “Mammal,” as used herein, refers to an individual belonging to the class Mammalia and includes, but not limited to, humans, domestic and farm animals, zoo animals, sports and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees, apes, and humans. In some embodiments, the subject is a human. However, in some embodiments, the subject is not a human.

[0042] As used herein, the term “treatment” refers to an intervention made in response to a disease, disorder or physiological condition manifested by a patient. The aim of treatment may include, but is not limited to, one or more of the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and the remission of the disease, disorder or condition. The term “treat” and “treatment” includes, for example, therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces one or more symptoms of the disorder and / or underlying risk factors. In some embodiments, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures.Those in need of treatment include those already affected by a disease or disorder or undesired physiological condition as well as those at a risk of developing the disease or disorder, and those in which the disease or disorder or undesired physiological condition is to be prevented. For example, in some embodiments treatment may enhance or reduce the level of serotonin in the subject, thereby to reduce, alleviate, or eradicate the symptom(s) of the disease(s). As used herein, the term “prevention” refers to any activity that reduces the burden of the individual later expressing those serotonin-related disease symptoms. This can take place at primary, secondary and / or tertiary prevention levels, wherein: a) primary prevention avoids the development of symptoms / disorder / condition; b) secondary prevention activities are aimed at early stages of the condition / disorder / symptom treatment, thereby increasing opportunities for interventions to prevent progression of the condition / disorder / symptom and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already establishedcondition / disorder / symptom by, for example, restoring function and / or reducing any condition / disorder / symptom or related complications. The term “prevent” does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.

[0043] As used herein, the term “effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results. As used herein, a “therapeutically effective amount” of a compound is an amount sufficient to provide any therapeutic benefit in the treatment or management of a disorder (e.g., a neuron mediated disorder or an ocular disorder), or to delay or minimize one or more symptoms associated with a disorder (e.g., a neuron mediated disorder or an ocular disorder). A therapeutically effective amount of an agent (e.g., a light-sensitive protein) refers to an amount of the agent, alone or in combination with one or more other therapies and / or therapeutic agents that provide any therapeutic benefit in the treatment or management of a disorder (e.g., a neuron mediated disorder or an ocular disorder). The term “therapeutically effective amount” can encompass an amount that alleviates a neuron mediated disorder or ocular disorder, improves or reduces the neuron mediated disorder or the ocular disorder, improves overall therapy, or enhances the therapeutic efficacy of another therapeutic agent.

[0044] As used herein, the term “blindness” refers to total or partial loss of vision. The blindness can be caused by, for example, degeneration or non-functioning of photoreceptors caused by any diseases and conditions (e.g., physical injuries). In some embodiments, the blindness is associated with conditions, such as, age-related macular degeneration or retinitis pigmentosa, glaucoma, late stage diabetic retinopathy, hereditary optic neuropathies, optic nerve injuries, and any combination thereof.

[0045] As used herein, the term “vision” refers to the ability of a subject to detect light as a stimulus for differentiation or action. Vision is intended to encompass the following: (1) light detection or perception, that is the ability to discern whether or not light is present; (2) light projection, that is the ability to discern the direction from which a light stimulus is coming; (3) resolution, that is the ability to detect differing brightness levels (i.e. , contrast) in a grating or letter target; and (4) recognition, that is the ability to recognize the shape of a visual target by reference to the differing contrast levels within the target. Thus, “vision” encompasses the ability to simply detect the presence of light (for example red light), including light having a wavelength between about 365 nm and about 700 nm, between about 530 nm and about 640 nm. In some embodiments, transfection of retinal neurons with a nucleic acid molecule (e.g. vector) encoding a light-sensitive protein disclosed herein provides retinal neurons, for example bipolar cellsand / or ganglion cells, with photosensitive membrane channels. Thus, it is possible to measure, with a light stimulus, the transmission of a visual stimulus to the animal's visual cortex, the area of the brain responsible for processing visual signals which constitutes a form of vision.

[0046] As used herein, the term “retinal cell” can refer herein to any of the cell types that comprise the retina, such as retinal ganglion cells; amacrine cells; horizontal cells; bipolar cells; and photoreceptor cells including rods and cones.

[0047] As used herein, the terms “light sensitivity” and “photosensitivity” are used interchangeably and refer to a notable or increased reactivity to light.

[0048] The term “AAV” or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses.

[0049] "AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector"). The term “AAV” includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV- 3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. See, e.g., Mori et al. (2004) Virology 330:375. The term “AAV” also includes chimeric AAV. “Primate AAV” refers to AAV isolated from a primate, “non-primate AAV” refers to AAV isolated from a non-primate mammal, “bovine AAV” refers to AAV isolated from a bovine mammal (e.g., a cow), etc. In some cases, the AAV may be AAV-2 or a derivative thereof.

[0050] An "rAAV vector" as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell. In general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0051] An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vectorparticle" or simply an "rAAV vector". Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.NUCLEIC ACIDS ENCODING ChRmine VARIANTS

[0052] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a channelrhodopsin (ChRmine) variant comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to SEQ ID NO:1 and comprising one or more of the following substitutions relative to SEQ ID NO:1 : i. an amino acid substitution of the alanine at amino acid 81 (A81); ii. an amino acid substitution of the leucine at position 47 (L47); ill. an amino acid substitution of the glutamine at position 130 (Q130); iv. an amino acid substitution of the threonine at position 119 (T119); and v. an amino acid substitution of the serine at position 35 (S35).

[0053] wherein numbering of the amino acid substitutions present in the ChRmine variants is with reference to the numbering of the amino acid residues in SEQ ID NO:1.

[0054] In some cases, the encoded ChRmine variants may be post-translationally modified to remove the N-terminal methionine. In some cases, the encoded ChRmine variants may be post-translationally modified such that the alanine at position 2 is acetylated. Thus, in some cases, as compared to SEQ ID NO:1 , the ChRmine variants do not include an N-terminal M, include an Ac-A at position 2, or both.

[0055] In some cases, as compared to SEQ ID NO:1 , the encoded ChRmine variants do not include an N-terminal M and include an Ac-A at position 2. While the first amino acid of these ChRmine variants is Ac-A, when numbered based on the numbering of SEQ ID NO:1 , it is still referred to as position 2.

[0056] The ChRmine variant of the present disclosure include seven transmembrane helices and a retinal chromophore covalently bound through a Schiff base to a conserved lysine residue. The ChRmine variant of the present disclosure are activatable by light and generate a photocurrent upon activation.

[0057] In certain cases, a channelrhodopsin variant of the present disclosure may include up to 20% difference in amino acid sequence relative to SEQ ID NO:1. The source of this difference may be substitutions and / or deletions. These substitutions may be conservative substitutions that do not affect the three-dimensional structure of the variant such that variantretains its properties. The location of the substitutions may be in regions of channelrhodopsin not required for, e.g., magnitude of photocurrent generated upon activation. Other properties include kinetics of channel closing, kinetics of channel opening, sensitivity, expression level, stability, etc. Such regions may be identified by aligning channelrhodopsin sequences from different sources. The deletions, if present, may be at the N-terminus, C-terminus, or both.

[0058] The ChRmine variants of the present disclosure has improved properties as compared to a wild-type channelrhodopsin (wt-ChRmine) having the amino acid sequence set forth in SEQ ID NO:1. For example, a channelrhodopsin variant provided herein as compared to wt-ChRmine, may have: (i) higher photocurrent such as, 0.5X or more, 1 X or more, 1.3X or more, 1.5X or more, 2X or more; (ii) higher sensitivity such as, 0.3X or more, 0.5 X or more, or 0.8X or more; (iii) slower closing; (iv) faster opening; (v) decreased desensitization; and / or (vi) faster recovery from inactivation, as compared to a wt-ChRmine having the amino acid sequence set forth in SEQ ID NO:1.

[0059] In particular, reduced light-dependent desensitization (also termed photocurrent desensitization herein) can be an increased stationary-peak-ratio or in particular an increased mean stationary-peak-ratio. A channelrhodopsin variant showing reduced light-dependent desensitization as described herein may provide a stationary-peak-ratio, or in particular a mean stationary-peak-ratio, that is higher than the stationary-peak-ratio, or in particular the mean stationary-peak-ratio, provided by wt-ChRmine.

[0060] In certain examples, an encoded channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitution at A81. In some cases, the substitution is A81S, A81T, A81 N, or A81Q. In some cases, the substitution may be A81S. This channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine having the amino acid sequence set forth in SEQ ID NO:1 : higher photocurrent (1.34x wild-type ChRmine), higher sensitivity (0.88x), significantly slower closing (1.34x), faster opening (0.73x), and significantly decreased desensitization (0.63 steady-state to peak current ratio).

[0061] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 andcomprise the amino acid substitution at L47. In some cases, the substitution is L47V, L47A, L47I, L47M, L47F, L47Y, or L47W. In some cases, the substitution may be L47V. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine: higher photocurrent (1.34x wild-type ChRmine), higher sensitivity (0.88x), significantly slower closing (1.34x), faster opening (0.73x), and significantly decreased desensitization (0.63 steady-state to peak current ratio).

[0062] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitution at Q130. In some cases, the substitution is Q130N, Q130S, or Q130T. In some cases, the substitution may be Q130N. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt- ChRmine: higher photocurrent (2.13x), higher sensitivity (0.89x), significantly slower closing (1.69x), faster opening (0.85x), and significantly decreased desensitization (0.73 steady-state to peak current ratio).

[0063] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitution at T119. In some cases, the substitution is T119A, T119V, T119I, T119L, T119M, T119F, T119Y, or T119W. In some cases, the substitution may be T119A. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine: significantly faster closing (0.67x), faster opening (0.86x), and significantly increased desensitization (0.03 steady-state to peak current ratio).

[0064] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitution at S35. In some cases, the substitution is S35V, S35A, S35I, S35L, S35M, S35F, S35Y, or S35W. In some cases, the substitution may be S35V. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine: higher sensitivity (0.90x), slower closing (1.33x), and decreased desensitization (0.49 steady-state to peak current ratio).

[0065] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitutions at L47 and T119. In some cases, the substitutions are L47V T119A. The channelrhodopsin variant may have increased desensitization (0.41 steadystate to peak current ratio) as compared to wt-ChRmine.

[0066] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitutions at Q130 and T119. In some cases, the substitutions are Q130N T119A. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine: significantly higher photocurrent (2.85x), significantly higher sensitivity (0.76x), slower closing (1.15x), and significantly faster opening (0.71x).

[0067] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitutions at A81 and T119. In some cases, the substitutions are A81S and T119A. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine: significantly higher photocurrent (2.40x), higher sensitivity (0.82x), faster opening (0.90x), and significantly increased desensitization (0.11 steady-state to peak current ratio).

[0068] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitutions at A81 , T119, and S35. In some cases, the substitutions are A81S, T119A, and S35V. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine: significantly higher photocurrent (2.03x), significantly higher sensitivity (0.74x), significantly faster closing (0.78x), significantly faster opening (0.78x), and significantly increased desensitization (0.09 steady-state to peak current ratio).

[0069] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitutions at Q130, T119, and S35. In some cases, the substitutions are Q130N, T119A, and S35V. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine: significantly higher photocurrent (2.13x) and higher sensitivity (0.90x).

[0070] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitutions at L47, T119, and S35. In some cases, the substitutions are L47V, T119A, and S35V. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine: significantly higher photocurrent (2.13x), significantly lower sensitivity (1.48x wild-type), significantly slower closing (1.46x), significantly increased desensitization (0.14 steady-state to peak current ratio), and significantly slower recovery from inactivation (0.85 paired pulse ratio).

[0071] In certain examples, a channelrhodopsin variant provided herein may include an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprise the amino acid substitutions at L47, T119, S35, and A81. In some cases, the substitutions are L47V, T119A, A81S, and S35V. The channelrhodopsin variant may have one or more of the following differences in a property as compared to wt-ChRmine: significantly higher photocurrent (2.77x), significantly higher sensitivity (0.69x), significantly slower closing (1.43x), faster opening (0.81x), significantly increased desensitization (0.25 steady-state to peak current ratio), and significantly slower recovery from inactivation (0.87 paired pulse ratio).

[0072] In addition to the amino acid substitution at one or more of the positions disclosed herein, a channelrhodopsin variant may include one or more of the following substitutions relative to SEQ ID NO:1 : T218, S220, Y260F, R136H, S138W, Y156F, and Y116F. In other aspects, in addition to the amino acid substitution at one or more of the positions disclosed herein, a channelrhodopsin variant may not include one or more of the following substitutions relative to SEQ ID NO:1 : T218, S220, Y260F, R136H, S138W, Y156F, and Y116F.

[0073] In certain aspects, the nucleic acid comprises a nucleotide sequence having at least 85% identity (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) to the nucleotide sequence of one of: SEQ ID NOs: 14-25.

[0074] The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO: 14 and the channelrhodopsin variant may comprise the amino acid substitution A81S. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO: 15 and the channelrhodopsin variant may comprise the amino acid substitution L47V. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO: 16 and the channelrhodopsin variant may comprise the amino acid substitution Q130N. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO: 17 and the channelrhodopsin variant may comprise the amino acid substitution S35V. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO: 18 and the channelrhodopsin variant may comprise the amino acid substitution T119A. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO: 19 and the channelrhodopsin variant may comprise the amino acid substitutions L47V T119A. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NQ:20 and the channelrhodopsin variant may comprise the amino acid substitutions Q130N T 119A. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO:21 and the channelrhodopsin variant may comprise the amino acid substitutions A81S T119A. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO:22 and the channelrhodopsin variant may comprise the amino acid substitution A81S T119A S35V. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO:23 and the channelrhodopsin variant may comprise the amino acid substitutions Q130N T119A S35V. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO:24 and the channelrhodopsin variant may comprise the amino acid substitutions L47V T119A S35V. The nucleotide sequence may have at least 85% identity to the nucleotide sequence of SEQ ID NO:25 and the channelrhodopsin variant may comprise the amino acid substitutions L47V T119A S35V A81S.

[0075] The nucleic acid comprising the nucleotide sequence encoding a ChRmine variant as described herein may be further include nucleotide sequence encoding a trafficking signal for exporting and / or targeting of the ChRmine variant to the cell membrane of a cell expressing the ChRmine variant For example, the ChRmine variant may be expressed as afusion with a trafficking sequence, a signal sequences, endoplasmic reticulum export sequences, a Golgi export signal and / or a membrane targeting motif, etc. Any suitable Golgi export signal and / or a membrane targeting motif may be used. The trafficking signal may be fused to the N-terminus or C-terminus of ChRmine variant. For example, the trafficking signal may be fused to the C-terminus of the ChRmine variant.

[0076] In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal can comprise an amino acid sequence having at least 90% or at least 95% sequence identity to KSRITSEGEYIPLDQIDINV (SEQ ID NO:38). In some embodiments, the trafficking signal can be derived from the amino acid sequence of a leucine-rich signal peptide sequence (Lucy tag). In some embodiments, the trafficking signal can comprise an amino acid sequence having at least 90% or at least 95% sequence identity to MRPQILLLLALLTLGLA (SEQ ID NO:39).

[0077] In some cases, the trafficking signal can be linked to the ChRmine variant sequence by a linker which can comprise any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker may be a cleavable linker.

[0078] In some cases, the nucleic acid may further include a nucleotide sequence encoding a targeting motif for targeting the ChRmine to a particular location in a cell. In some cases, the targeting motif may target the ChRmine variant to the soma of a neuron. In some cases, the soma targeting motif can comprise an amino acid sequence having at least 85%, at least 90% or at least 95% sequence identity to QSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVIDMRSMSSIDSFISCATDFPEATR FFCYENEV (SEQ ID NQ:40).

[0079] The nucleotide sequence encoding a subject channelrhodopsin variant can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow expression of the nucleotide sequence in a recombinant cell that is genetically modified to produce the channelrhodopsin variant.

[0080] Suitable promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus;mouse metallothionein-l promoter; and the like. Additional promoters suitable for controlling expression of a ChRmine variant of the present disclosure include a gamma-synuclein (SNCG) promoter (e.g., Chaffiol et al. (2017) Mol. Ther. 25(11) 2546), a CBh promoter (e.g., Grey et al. (2011) Hum. Gene Ther. 22(9): 1143-53), a miniCAG promoter (e.g., Grey et al. (2011) Hum. Gene Ther. 22(9): 1143-53), a neurofilament heavy (NEFH) promoter (Millington- Ward et al. (2020) Sci. Rep. 10:16515), a G protein-coupled receptor kinase 1 (GRK1) promoter (e.g., Khani et al. (2007) Invest. Ophthalmol. Vis. Sci. 48(9):3954-61), a retinaldehyde-binding protein 1 (RLBP1) promoter (e.g., Choi et al. (2015) Mol. Ther. Methods Clin. Dev. 2: 15022; Vogel et al. (2007) Invest. Ophthalmol. Vis. Sci. 48, 3872-3877), a vitelliform muscular dystrophy-2 (VMD2) promoter (e.g., Conlon et al. (2013) Hum. Gene Ther. Clin. Dev. 24, 23-28), a synapsin I (Syn1) promoter (e.g., Kugler et al. (2003)), an enhSynl promoter (e.g., Hioki et al. (2007) Gene Ther.14(11):872-82), or a functional fragment or variant thereof.

[0081] In some cases, the nucleic acid comprising the nucleotide sequence encoding ChRmine variant may further include the nucleotide sequence encoding a Golgi export signal, where the nucleotide sequence encoding the channelrhodopsin variant is a first nucleotide sequence and the nucleotide sequence encoding the Golgi export signal is a second nucleotide sequence, where the first nucleotide sequence is upstream of and in frame with the second nucleotide sequence and the channelrhodopsin variant is fused at the C-terminus to the Golgi export signal.

[0082] In some cases, the nucleic acid comprising a first nucleotide sequence encoding ChRmine variant and a second nucleotide sequence encoding the Golgi export signal may further include a third nucleotide sequence encoding a SOMA targeting motif, where the third nucleotide sequence is downstream to and in frame with the second nucleotide sequence and the SOMA targeting motif is fused to the C-terminus of the Golgi export signal.

[0083] A nucleotide sequence encoding a subject channelrhodopsin variant can be present in an expression vector and / or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1 , pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia). Expression vectors generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion of nucleic acid sequences encoding polypeptides. A selectable marker operative in the expression host cell may be present.

[0084] Nucleic acids, e.g., as described herein, may, in some instances, be introduced into a cell, e.g., by contacting the cell with the nucleic acid. Cells with introduced nucleic acids will generally be referred to herein as genetically modified cells. Various methods of nucleic acid delivery may be employed including but not limited to e.g., naked nucleic acid delivery, viral delivery, chemical transfection, biolistics, and the like.

[0085] The nucleic acids of the present disclosure may be provided in a kit. The kit may include additional components such as reconstitution buffer for resuspending the nucleic acid provided in the kit in a lyophilized form.

[0086] The nucleic acid can be a recombinant expression vector, for example, a viral vector. Examples of viral vector include, but are not limited to, adeno-associated viral vectors, lentiviral vectors, herpes simplex virus vectors, and retroviral vectors. In the nucleic acid, the nucleotide sequence encoding the channelrhodopsin variant is operably linked to a transcriptional control element. It can be advantageous in some embodiments that the transcriptional control element is functional in a photoreceptor cell. The photoreceptor cell can be a rod cell, a cone cell, a retinal cell, or a combination thereof. The transcriptional control element can be, for example, promoter (e.g., a retinal cell-specific promoter). Non-limiting examples of the promoter include synapsin promoter, a CAG promoter, a cytomegalovirus promoter (CMV) promoter, a grm6 promoter, a Pleiades promoter, a ChAT promoter, a V-glut promoter, a GAD promoter, a PV promoter, a somatostatin (SST) promoter, a neuropeptide Y (NPY) promoter, a VIP promoter, a red cone opsin promoter, rhodopsin promoter, a rhodopsin kinase promoter, vitelliform macular dystrophy 2 (VMD2) gene promoter, an interphotoreceptor retinoid-binding protein (IRBP) gene promoter, elongation factor- 1 alpha (EF-1 alpha) promoter, and a combination thereof.

[0087] The nucleic acids comprising a coding sequence for the channelrhodopsin variants can be used for treating or ameliorating blindness, restoring or enhancing vision and photosensitivity, treating or ameliorating vision loss in a subject. In some embodiments, the method comprises delivering (e.g., injecting) the nucleic acid molecule into the thalamus, e.g., the lateral geniculate nucleus (LGN) of the subject. The expression of the channelrhodopsin variant can be controlled by a transcription regulatory element, for example a promoter selected from the group of Human elongation factor-1 alpha (EF-1 alpha), Human cytomegalovirus promoter (CMV) or CAG promoter. Also disclosed include a composition, for example apharmaceutical composition, comprising the nucleic acid molecule (e.g., a vector) comprising the coding sequence for the channelrhodopsin variant.

[0088] Many different viral and non-viral vectors and methods of their delivery, for use in gene delivery (including gene therapy), are known, including adenovirus vectors, adeno- associated virus (AAV) vectors, retrovirus vectors, lentiviral vectors, herpes virus vectors, liposomes, poxviruses, naked DNA administration, plasmids, cosmids, phages, encapsulated cell technology, and the like.

[0089] In certain cases, an AAV virus may include a capsid that infects retinal cells, e.g., neurons. The capsid may be naturally occurring or modified for increased specificity and / or increased infectivity.ChRmine Variants

[0090] A ChRmine variant is provided, where the ChRmine variant comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 92%, at least 95% at least 96% at least 97%, at least 98%, at least 99% sequence identity) to the amino acid sequence set forth in SEQ ID NO:1 and comprising one or more of the following substitutions relative to SEQ ID NO:1 :

[0091] an amino acid substitution of the alanine at amino acid 81 (A81);

[0092] an amino acid substitution of the leucine at position 47 (L47);

[0093] an amino acid substitution of the glutamine at position 130 (Q130); and

[0094] an amino acid substitution of the threonine at position 119 (T119);

[0095] an amino acid substitution of the serine at position 35 (S35), and where the numbering of the positions is based on the numbering of SEQ ID NO:1, optionally, where relative to SEQ ID NO:1, the channelrhodopsin variant does not include an N-terminus Methionine and comprises an acetyl-alanine at position 2, based on the numbering of SEQ ID NO:1

[0096] As described in the preceding section, the amino acid substitution at A81 is A81S, A81T, A81 N, or A81Q; the amino acid substitution at L47 is L47V, L47A, L47I, L47M, L47F, L47Y, or L47W; the amino acid substitution at Q130 is Q130N, Q130S, or Q130T; the amino acid substitution at T119 is T119A, T119V, T119I, T119L, T119M, T119F, T119Y, or T119W; and / or the amino acid substitution at S35 is S35V, S35A, S35I, S35L, S35M, S35F, S35Y, or S35W, numbered according to SEQ ID NO:1.

[0097] The channelrhodopsin variant may comprise one of the following double substitutions, triple substitutions, or quadruple substitution:

[0098] L47 T119A;

[0099] Q130N T119A;

[0100] A81S T119A;

[0101] A81S T119A S35V;

[0102] Q130N T119A S35V;

[0103] L47V T119A S35V; and

[0104] L47V T119A S35V A81S.

[0105] The amino acid sequence of the channelrhodopsin variant may have at least85%, at least 90%, at least 92%, at least 95% at least 96% at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:1.

[0106] The channelrhodopsin variant may comprise the amino acid sequence set forth in any one of SEQ ID NOs:26-37. When numbered based on any of SEQ ID NOs: 26-37, the position of the amino acid substitutions shifts by -1 since SEQ ID NO: 26-37 do not include the N-terminal M present in SEQ ID NO:1.

[0107] Disclosed herein includes methods and compositions for expressing one or more of the channelrhodopsin variant disclosed herein in cells, tissues, organs, and / or subjects, where the channelrhodopsin variant can be activated by contact with one or more pulses of light, which results in strong depolarization of the cells or the cells in the tissues, organs and / or subjects. In some embodiments, the expression of the channelrhodopsin variant is used to control cells, tissues, organs, and / or subjects in vivo, ex vivo, and / or in vitro in response to pulses of light of a suitable wavelength. A cell, comprising (a) a channelrhodopsin variant, (b) a nucleic acid molecule comprising a coding sequence of the channelrhodopsin variant, or both, is provided. The cell can be, for example, a mammalian cell or non-mammalian cell. In some embodiments, the cell is a rod cell, a cone cell, or a retina cell. The cell can be a neuronal cell, an electrically active cell, or both. In some embodiments, the cell is a recombinant host cell, for example, a mammalian cell, a bacterial cell, a yeast cell, an insect cell, a plant cell, or a combination thereof. Some embodiments provided a composition comprising one or more of (a) the cell, (b) the channelrhodopsin variant, and (c) the nucleic acid molecule comprising the coding sequence of the channelrhodopsin variant. The composition can be, for example, a pharmaceutical composition comprising one or more pharmaceutically acceptable excipient.The pharmaceutically acceptable excipient can include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween™, Pluronics™ or polyethylene glycol (PEG).HOST CELLS

[0108] The present disclosure provides isolated genetically modified cells (e.g., in vitro cells, ex vivo cells, cultured cells, neurons, etc.) that are genetically modified with a subject nucleic acid. In some aspects, a subject isolated genetically modified cell can produce a subject channelrhodopsin variant. In some instances, a genetically modified cell may be used in the screening, etc.

[0109] Suitable cells include eukaryotic cells, such as a mammalian cell, an insect cell, a yeast cell; and prokaryotic cells, such as a bacterial cell. Introduction of a subject nucleic acid into the host cell can be affected, for example by calcium phosphate precipitation, DEAE dextran mediated transfection, liposome-mediated transfection, electroporation, viral infection, or other known methods.KITS

[0110] Aspects of the present disclosure include kits. The kit may also include one or more components, such as, a nucleic acid as described herein, a channelrhodopsin variant as described herein, a cell as described herein, and a composition as described herein. The components may be present in separate containers. The kit may include instructions for assaying channelrhodopsin variant property, such as, the properties disclosed herein. A channelrhodopsin variant property may be measured using a suitable method, such as, the methods disclosed herein.METHODS

[0111] The channelrhodopsin variants of the disclosure may be used in any way that channelrhodopsins have been used. For example, they may be expressed in a retinal andcontacted with light to activate the channelrhodopsin variant and generate a photocurrent. The retinal cell may be a neuron.

[0112] The channelrhodopsin variants may be used in optogenetics. Accordingly, certain embodiments of the disclosure provide an optogenetic method comprising genetically modifying a subject to express in the subject’s retina a channelrhodopsin variant disclosed herein, applying stimulating light to the subject’s brain, and imaging the subject’s brain. A subject can be a mammal, e.g., a human, a non-human primate, a bovine, a porcine, a feline, or a canine animal. The details of the optogenetic methods are well known in the art and generally applying such methods using a channelrhodopsin variant disclosed herein is within the purview of the disclosure.

[0113] The channelrhodopsin variants disclosed herein can be used to treat or prevent neuronal disorders, ocular disorders, or both. In some embodiments, the channelrhodopsin variant can be used to restore and / or improve light sensitivity and / or vision of a subject. The visual signal is initially processed in the retina and most of conscious vision is then relayed to the lateral geniculate nucleus (LGN) of the thalamus, which in turn projects to the primary visual cortex. Since the visual signal is processed less in the LGN than in the cortex, and the number of cells dedicated to the same visual angle or retinal area is smaller in the LGN than in the cortex, it is contemplated herein that, in some embodiments, the cells of the LGN can be stimulated to restore vision. For example, LGN cells can be activated optogenetically using a composition (e.g., an expression vector, including a viral vector) comprising a coding sequence for a channelrhodopsin variant to illuminate with visual patterns the axon terminals of LGN cells where they form connections with the visual cortex, in the more accessible surface of the brain. LGN cell stimulation can, in some embodiments, evoke meaningful responses in blind and / or normal-sighted subjects. For example, normal or blind subjects can be caused to express one or more of the channelrhodopsin variants in the LGN cells (e.g., via an AAV vector).

[0114] In some embodiments, administering to the subject the recombinant expression vector comprises injecting the vector into the retain or brain (e.g., lateral geniculate nucleus) of the subject. In some embodiments, the subject is provided with a visual prosthesis before, at the same time as, or after delivery of said vector. In some embodiments, the visual prosthesis is a retinal implant, a cortical implant, a lateral geniculate nucleus implant, or an optic nerve implant.

[0115] The channelrhodopsin variant or a cell comprising same as disclosed herein can be used in a high-throughput screenings, in particular in drug discovery. A high-throughput screening (HTS), is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. HTS allows a researcher to effectivelyconduct millions of biochemical, genetic or pharmacological tests in a short period of time, often through a combination of modern robotics, data processing and control software, liquid handling devices, and sensitive detectors. By this process, one may rapidly identify active agents which modulate a particular biomolecular pathway; particularly a substance modifying a cellular function that is governed by the voltage potential of the cell.

[0116] Briefly, HTS uses an approach to collect a large amount of experimental data on the effect of a multitude of substances on a particular target in a relatively short time. A screen, in this context, is the larger experiment, with a single goal (usually testing a scientific hypothesis), to which all this data may subsequently be applied. For HTS cells according to the invention may be used in a tissue plate, such as a multi well plate, e.g. a 96-well plate. Then the cell in the plate is contacted with the test substance for a time sufficient to interact with the channelrhodopsin variant. The test substance may be different from well to well across the plate. After incubation time has passed, measurements are taken across all the plate's wells, either manually or by a machine and optionally compared to measurements of a cell which has not been contacted with the test substance. Manual measurements may be necessary when the researcher is using patch-clamp, looking for effects not yet implemented in automated routines. Otherwise, a specialized automated analysis machine can run a number of experiments on the wells (such as analyzing light of a particular frequency or a high-throughput patch-clamp measurement). Depending upon the results of this first assay, the researcher can perform follow up assays within the same screen by using substances similar to those identified as active (i.e. modifying an intracellular cyclic nucleotide level) into new assay plates, and then re-running the experiment to collect further data, optimize the structure of the chemical agent to improve the effect of the agent on the cell. Automation is an important element in HTS's usefulness. A specialized robot is often responsible for much of the process over the lifetime of a single assay plate, from creation through final analysis. An HTS robot can usually prepare and analyze many plates simultaneously, further speeding the data- co 11 ection process. Examples for apparatuses suitable for HTS in accordance with the present invention comprise a Fluorometric Imaging Plate Reader (FLIPRTM; Molecular Devices), FLEXstationTM (Molecular Devices), Voltage Ion Probe Reader (VI PR, Aurora Biosciences), Attofluor® Ratio Vision® (ATTO). Various optogenetic approaches to drug discovery, in particular in neuroscience, are generally known in the art.

[0117] Thus, the presently disclosed mutant ion channel is particularly useful as a research tool, such as in a non-therapeutic use for light-stimulation of electrically excitable cells, in particular neurons.EXAMPLES

[0118] The following examples are offered to illustrate, but not to limit any embodiments provided by the present disclosure.Example 1 : structure-based protein engineering

[0119] While many channelrhodopsins have been characterized, our ability to control neural electrical signals with high spatial and temporal fidelity in a minimally invasive manner is still limited by intrinsic biophysical properties of reported channelrhodopsins.

[0120] To address this limitation, we used structure-based protein engineering to design a series of novel channelrhodopsin variants to serve as ultrapotent optogenetic actuators of cellular electrical activity. These channelrhodopsins show significantly increased photocurrent magnitude and light sensitivity over currently available tools. These engineered variants expand the scale and scope of applied optogenetics and are particularly useful for therapeutic applications.

[0121] We developed a procedure for capturing structures of channelrhodopsin in light- stimulated states by cryo-EM in which particles are illuminated on cryo-EM grids during the plunge freezing process. A fiber optic cable connected to an LED light engine is positioned with its output inside of the temperature and humidity-controlled chamber of a Vitrobot Mark IV. The fiber was fixed at the bottom of the chamber to create a beam of light in the plunge path of a cryo-EM grid with center wavelength of 550nm, diameter of 8-10 mm, and intensity of 5-7.3 mW / mm2. After application of particles to the grid and blotting to remove excess liquid, the grid is plunged through the light beam. This results in exposure of the particles on the grid to light for ~6 ms at supersaturating intensity. Particles are dark for ~30ms following light exposure before vitrification in liquid ethane. Given a closing time constant of ~60 ms for wild-type ChRmine at 32° C, this is expected to capture most particles in a light-stimulated state.

[0122] We purified ChRmine, reconstituted it in lipid nanodiscs, dark-adapted samples prior to grid preparation, and light stimulated the sample during plunge-freezing. A cryo-EM dataset collected on this sample resulted in a reconstruction to an overall resolution of 2.35 A. The experimental map shows conformational changes at the Schiff base and retinal moiety consistent with isomerization from all-trans in the dark state to 13-cis retinal in the light stimulated state. To account for the possibility that some particles relax to an all-trans configuration in the interval between light stimulation and vitrification, we refined a model with mixed occupancy of all-trans and 13-cis retinal. From initial occupancies of 0.5, refinement yielded a model with predominantly 13-cis retinal in all three ChRmine subunits (0.68, 0.97, and0.92 fractional 13-cis retinal in subunits A-C, respectively). Analysis of the new structure provided insight into regions that remain constricted in the excited state. We reasoned targeting these sites with mutations that alter size and polarity of the constrictions would increase channel photocurrents and light sensitivity (Fig. 1).

[0123] We designed a panel of variants based on this structural analysis. While mutations were predicted to increase photocurrents and sensitivity, changes in these properties is often correlated with changes in kinetic properties in other channelrhodopsins18. For example, increased photocurrent often slows channel closing. We therefore designed protocols to systematically test multiple channel properties and compare them to wild-type ChRmine (Figs. 2-7, Table 1). Peak photocurrent magnitude was measured in response to saturating 5 ms duration teal (510nm) light stimulation and was 2.1 ± 0.3 nA (all values are mean ± sem) for wildtype ChRmine (Fig. 2). Light sensitivity was determined from steps of 5 ms duration teal (510nm) light stimulation of varying power (Fig. 3) and normalized to sensitivity of wild-type ChRmine. Closing and opening rates are reported as time constants from single exponential fits to 5 ms duration teal (510nm) light stimulation and were 48.8 ± 2.2 ms and 4.6 ± 0.4 ms for wildtype ChRmine (Figs. 4-5). Channel desensitization was measured during prolonged teal light stimulation (500 ms) and reported as the ratio of steady state (end of pulse) to peak current.ChRmine currents decrease 65% over 500ms, giving a steady-state to peak current ratio of 0.35 ± 0.02. Finally, recovery from channel inactivation was measured using paired 5 ms teal light stimuli 165 ms apart and is reported as the ratio of peak currents elicited by the pulses.ChRmine recovers nearly completely in the 165 dark interval between stimuli, showing 0.94 ± 0.01 fractional recovery of first stimulus current.

[0124] Of sixteen single mutants evaluated, five showed the most promising differences in properties compared to wild-type ChRmine. Four mutants (A81S, L47V, Q130N, T119A) were designed to alter steric and electrostatic properties of channel constrictions remaining in the light-stimulated structure. The fifth (S35V) was predicted to increase functional expression and protein stability by reducing polarity of the channel surface exposed to the apolar lipid membrane. Subsequently, we evaluated combinatorial effects of these mutations. Two double mutants, three triple mutants, and one quadruple mutant showed significant improvements in channel properties for optogenetic applications. Significant differences in channel properties are indicated in Figs. 2-7 and summarized in Table 1. A summary of notable differences follows below.

[0125] A81S (ChRmineA; variant 1) showed higher photocurrent (1.34x wild-type ChRmine), higher sensitivity (0.88x), significantly slower closing (1.34x), faster opening (0.73x),significantly decreased desensitization (0.63 steady-state to peak current ratio), and similar recovery from inactivation (0.97 paired pulse ratio).

[0126] L47V (ChRmineL; variant 2) showed higher photocurrent (2.00x), significantly higher sensitivity (0.60x), significantly slower closing (2.26x), significantly faster opening (0.63x), significantly decreased desensitization (0.77 steady-state to peak current ratio), and similar recovery from inactivation (0.98 paired pulse ratio).

[0127] Q130N (ChRmineQ; variant 3) showed higher photocurrent (2.13x), higher sensitivity (0.89x), significantly slower closing (1.69x), faster opening (0.85x), significantly decreased desensitization (0.73 steady-state to peak current ratio), and similar recovery from inactivation (1.0 paired pulse ratio).

[0128] S35V (ChRmineS; variant 4) showed similar photocurrent (1.09x), higher sensitivity (0.90x), slower closing (1.33x), similar opening (0.97x), decreased desensitization (0.49 steady-state to peak current ratio), and similar recovery from inactivation (0.97 paired pulse ratio).

[0129] T119A (ChRmineT; variant 5) showed similar photocurrent (1.19x), similar sensitivity (1.05x), significantly faster closing (0.67x), faster opening (0.86x), significantly increased desensitization (0.03 steady-state to peak current ratio), and significantly slower recovery from inactivation (0.80 paired pulse ratio).

[0130] L47V T119A (ChRmineLT; variant 6) showed similar photocurrent (1.12x), significantly lower sensitivity (1.88x), slower closing (1.31x), significantly slower opening rate (1.67x), increased desensitization (0.41 steady-state to peak current ratio), and similar recovery from inactivation (0.94 paired pulse ratio).

[0131] Q130N T119A (ChRmineQT; variant 7) showed significantly higher photocurrent(2.85x), significantly higher sensitivity (0.76x), slower closing (1.15x), significantly faster opening (0.71x), similar desensitization (0.32 steady-state to peak current ratio), and similar recovery from inactivation (0.91 paired pulse ratio).

[0132] A81S T119A (ChRmineAT; variant 8) showed significantly higher photocurrent (2.40x), higher sensitivity (0.82x), similar closing rate (0.94x), faster opening (0.90x), significantly increased desensitization (0.11 steady-state to peak current ratio), and significantly slower recovery from inactivation (0.84 paired pulse ratio).

[0133] A81S T119A S35V (ChRmineATS; variant 9) showed significantly higher photocurrent (2.03x), significantly higher sensitivity (0.74x), significantly faster closing (0.78x), significantly faster opening (0.78x), significantly increased desensitization (0.09 steady-state to peak current ratio), and significantly slower recovery from inactivation (0.85 paired pulse ratio).

[0134] Q130N T119A S35V (ChRmineQTS; variant 10) showed significantly higher photocurrent (2.13x), higher sensitivity (0.90x), similar closing rate (0.95x), similar opening rate (0.94x), similar desensitization (0.22 steady-state to peak current ratio), and similar recovery from inactivation (0.90 paired pulse ratio).

[0135] L47V T119A S35V (ChRmineLTS; variant 11) showed significantly higher photocurrent (2.13x), significantly lower sensitivity (1.48x wild-type), significantly slower closing (1.46x), similar opening (0.93x), significantly increased desensitization (0.14 steady-state to peak current ratio), and significantly slower recovery from inactivation (0.85 paired pulse ratio).

[0136] L47V T119A S35V A81S (ChRmineLTSA; variant 12) showed significantly higher photocurrent (2.77x), significantly higher sensitivity (0.69x), significantly slower closing (1.43x), faster opening (0.81x), significantly increased desensitization (0.25 steady-state to peak current ratio), and significantly slower recovery from inactivation (0.87 paired pulse ratio).

[0137] Among these variants, six (variants 7-12) are particularly promising for applied optogenetic applications. Compared to wild-type ChRmine, ChRmineQTS (variant 10) gives higher photocurrent. ChRmineQT (variant 7) gives higher photocurrent, higher sensitivity, and faster opening. ChRmineAT (variant 8) gives higher photocurrent and increased desensitization. ChRmineLTS (variant 11) gives higher photocurrent, lower sensitivity, slower closing, increased desensitization, and slower recovery from inactivation. ChRmineATS (variant 9) gives higher photocurrent, higher sensitivity, faster opening, increased desensitization, and slower recovery from inactivation. ChRmineLTSA (variant 12) gives higher photocurrent, higher sensitivity, slower closing, increased desensitization, and slower recovery from inactivation.

[0138] All are extremely potent, generating significantly larger photocurrents compared to wild-type ChRmine. This is exceptional as wildtype ChRmine is already among the most potent channelrhodopsins reported to date15’18’19. Similarly, the higher sensitivity observed in variants 7,9, and 12 is exceptional given wildtype ChRmine is already among the most sensitive channelrhodopsins known15’18’19. The variants described here outperform previously reported ChRmine variants including rsChRmine, hsChRmine, frChRmine, and ChReef15-17’20, particularly in photocurrent magnitude and light sensitivity. Other properties vary and the optimal channelrhodopsin variant can be selected based on requirements of the specific application.

[0139] Table 1 : Summary of channel properties.Values are mean ± sem with number of cells tested in parentheses. Statistically significant differences as assessed with Fishers LSD test are bold (*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001).

[0140] Fig. 1. Cryo-EM structures of ChRmine in dark and light-stimulated states reveal targets for structure-based engineering of channel properties, (a) Single subunit from light-stimulated and (b) dark-state (blue) ChRmine viewed from the membrane. Calculated channel pores are gray, (c) Pore radii from each structure versus probe distance traveledthrough channel pore. Evident constrictions are labeled, (d) Sliced view of central constriction and (e) intracellular constriction. Regions illustrated are boxed in (a). Constricting residues selected for targeted mutagenesis are shown as sticks.

[0141] Fig. 2. Photocurrents recorded from new channelrhodopsin variants.Maximum currents recorded from cultured cells in response to 5 ms, 510 nm light stimulation (left) and current magnitude normalized to average wild-type ChRmine current (right). Notable constructs are colored (dark grey=wild-type, green=single mutant, orange=double mutant, blue=triple mutant, red=quadruple mutant). Data is mean ± SEM with individual data points (each from a separate cell). Statistical differences to wild-type ChRmine assessed with Fishers LSD test. *P<0.05, *P<0.01, ***P<0.001, ****P<0.001.

[0142] Fig. 3. Light sensitivity of new channelrhodopsin variants. ED50 values, normalized to wild-type ChRmine, from fits to current versus light intensity recorded from successive sweeps with increasing intensity of 5ms 510nm light stimulation. Notable constructs are colored (dark grey=wild-type, green=single mutant, orange=double mutant, blue=triple mutant, red=quadruple mutant). Data is mean ± SEM with individual data points (each from a separate cell). Statistical differences to wild-type ChRmine assessed with Fishers LSD test.*P<0.05, *P<0.01, ***P<0.001, ****P<0.001.

[0143] Fig. 4. Closing rate recorded from new channelrhodopsin variants. Closing time constant from single exponential fits to current decay following 5 ms, 510 nm light stimulation (left) and values normalized to average wild-type ChRmine closing time constant (right). Notable constructs are colored (dark grey=wild-type, green=single mutant, orange=double mutant, blue=triple mutant, red=quadruple mutant). Data is mean ± SEM with individual data points (each from a separate cell). Statistical differences to wild-type ChRmine assessed with Fishers LSD test. *P<0.05, *P<0.01 , ***P<0.001, ****P<0.001.

[0144] Fig.5. Opening rate recorded from new channelrhodopsin variants. Opening time constant from single exponential fits to current increase during 5 ms, 510 nm light stimulation (left) and values normalized to average wild-type ChRmine opening time constant (right). Notable constructs are colored (dark grey=wild-type, green=single mutant, orange=double mutant, blue=triple mutant, red=quadruple mutant). Data is mean ± SEM with individual data points (each from a separate cell). Statistical differences to wild-type ChRmine assessed with Fishers LSD test. *P<0.05, *P<0.01 , ***P<0.001, ****P<0.001.

[0145] Fig. 6. Desensitization of channelrhodopsin variants. Ratio of steady state (recorded at end of pulse) and peak current during 500 ms, 510 nm light stimulation. Notable constructs are colored (dark grey=wild-type, green=single mutant, orange=double mutant,blue=triple mutant, red=quadruple mutant). Data is mean ± SEM with individual data points (each from a separate cell). Statistical differences to wild-type ChRmine assessed with Fishers LSD test. *P<0.05, *P<0.01, ***P<0.001 , ****P<0.001.

[0146] Fig. 7. Recovery from inactivation of channelrhodopsin variants. Ratio of peak currents recorded from paired 5 ms, 510 nm light stimulation with an inter-stimulus duration of 165 ms. Notable constructs are colored (dark grey=wild-type, green=single mutant, orange=double mutant, blue=triple mutant, red=quadruple mutant). Data is mean ± SEM with individual data points (each from a separate cell). Statistical differences to wild-type ChRmine assessed with Fishers LSD test. *P<0.05, *P<0.01 , ***P<0.001 , ****P<0.001.

[0147] Amino acid sequence of wildtype ChRmine from Rhodomonas lens, wt ChRmine fusion, golgi export signal, Mruby, SOMA targeting motif, the channelrhodopsin variants are listed below.WILDTYPE ChRmine:

[0148] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWT SRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY MLTCPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIF AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNW TEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:1)WILDTYPE CHRMINE 1 :309-LINKER-GQLGI EXPORT SIGNAL-LINKER-MRUBY-LINKER- SOMA TARGETING MOTIF:

[0149] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWT SRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY M LTC PM LVYD LLYQ LRAPYR VSCSAI I FA I LM SG VLAE FYA EG D P R LR N G AYA WYG FGC FWF I F AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNW TEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS-AAA-KSRITSEGEYIPLDQIDINV-VPV-MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMRIKVIEGGPLPFAFDILA TSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGVVTVMQDTSLEDGCLVYHVQVR GVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHMALKVDGGGHLSCSFVTTYRSKKT VGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFAGLGGGMDELYK-ALE-QSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVIDMRSMSSIDSFISCATDFPEATR FFCYENEV (SEQ ID N0:41)GOLGI EXPORT SIGNAL: KSRITSEGEYIPLDQIDINV (SEQ ID NO:38)SOMA TARGETING MOTIF:QSQPILNTKEMAPQSKPPEELEMSSMPSPVAPLPARTEGVIDMRSMSSIDSFISCATDFPEATR FFCYENEV (SEQ ID NO:40)A81S:

[0150] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWT SRGPSKRTSFYAGYQEEQNLALFVNFFSMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY M LTC PM LVYD LLYQ LRAPYR VSCSAI I FA I LM SG VLAE FYA EG D P R LR N G AYA WYG FGC FWF I F AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNW TEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:2)A81S:

[0151] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFY AGYQEEQNLALFVNFFSMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLL YQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAK SCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:26)L47V:

[0152] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYVGYESWT SRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY M LTC PM LVYD LLYQ LRAPYR VSCSAI I FA I LM SG VLAE FYA EG D P R LR N GAYA WYG FGC FWF I F AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNW TEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:3)L47V:

[0153] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYVGYESWTSRGPSKRTSFY AGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLL YQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:27)Q130N:

[0154] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWT SRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY MLTCPMLVYDLLYNLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIF AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNW TEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:4)Q130N:

[0155] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFY AGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLL YNLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAK SCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:28)S35V:

[0156] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWVCFIVLTITTFYLGYESWT SRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY M LTC PM LVYD LLYQ LRAPYR VSCSAI I FA I LM SG VLAE FYA EG D P R LR N G AYA WYG FGC FWF I F AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNW TEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:5)S35V:

[0157] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWVCFIVLTITTFYLGYESWTSRGPSKRTSFY AGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLL YQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAK SCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:29)T119A:

[0158] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY M LACPM LVYDLLYQLRAPYRVSCSAI I FAI LMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFI F AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:6)T119A:

[0159] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLL YQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAK SCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:30)L47V T119A:

[0160] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYVGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY M LACPM LVYDLLYQLRAPYRVSCSAI I FAI LMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFI F AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:7)L47V T119A:

[0161] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYVGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLL YQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAK SCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:31)Q130N T119A:

[0162] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY MLACPMLVYDLLYNLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIF AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:8)Q130N T119A:

[0163] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLL YNLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAK SCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:32)A81S T119A:

[0164] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFSMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY M LACPM LVYDLLYQLRAPYRVSCSAI I FAI LMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFI F AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:9)A81S T119A:

[0165] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFY AGYQEEQNLALFVNFFSMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLL YQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAK SCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:33)A81S T119A S35V:

[0166] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWVCFIVLTITTFYLGYESWT SRGPSKRTSFYAGYQEEQNLALFVNFFSMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADY M LACPM LVYDLLYQLRAPYRVSCSAI I FAI LMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFI F AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO: 10)A81S T119A S35V:

[0167] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWVCFIVLTITTFYLGYESWTSRGPSKRTSFY AGYQEEQNLALFVNFFSMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLL YQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:34)Q130N T119A S35V:

[0168] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWVCFIVLTITTFYLGYESWT SRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLLYNLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIF AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNW TEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRR LS (SEQ ID NO:11)Q130N T119A S35V:

[0169] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWVCFIVLTITTFYLGYESWTSRGPSKRTSFY AGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLL YNLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:35)L47V T119A S35V:

[0170] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHVWCFIVLTITTFYVGYESWT SRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIF AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNW TEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO: 12)L47V T119A S35V:

[0171] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWVCFIVLTITTFYVGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAK SCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:36)L47V T119A S35V A81S:

[0172] MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWVCFIVLTITTFYVGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFSMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIF AYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO: 13)L47V T119A S35V A81S:

[0173] Ac-AHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWVCFIVLTITTFYVGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFSMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLACPMLVYDLL YQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQY SRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS (SEQ ID NO:37)Nucleotide sequences encoding Channelrhodopsin variants (ChRmine variants) (underlined nucleotides denote the codon encoding the amino acid substitution):1. ChRmineA:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactggtcttgctttatcgtgctgacaatcaccacattctacctgggctatgagtcctggacatctcggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttcttttccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctgacctgcccaatgctggtgtacgatctgctgtatcagctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctcca tgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcc cactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID NO: 14) ChRmineL:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactggtcttgctttatcgtgctgacaatcaccacattctacgtgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttctttgccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctgacctgcccaatgctggtgtacgatctgctgtatcagctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctcca tgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcc cactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID NO: 15) ChRmineQ:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactggtcttgctttatcgtgctgacaatcaccacattctacctgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttctttgccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctgacctgcccaatgctggtgtacgatctgctgtataacctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctcca tgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcccactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID NO: 16) ChRmineS:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactgggtgtgctttatcgtgctgacaatcaccacattctacctgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttctttgccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctgacctgcccaatgctggtgtacgatctgctgtatcagctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctcca tgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcc cactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID NO: 17) ChRmineT:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactggtcttgctttatcgtgctgacaatcaccacattctacctgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttctttgccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctggcctgcccaatgctggtgtacgatctgctgtatcagctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctcca tgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcc cactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID NO: 18) ChRmineLT:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactggtcttgctttatcgtgctgacaatcaccacattctacgtgggctatgagtcctggacatctcggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttctttgccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctggcctgcccaatgctggtgtacgatctgctgtatcagctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctcca tgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcc cactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID NO: 19) ChRmineQT: atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactggtcttgctttatcgtgctgacaatcaccacattctacctgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttctttgccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctggcctgcccaatgctggtgtacgatctgctgtataacctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccGAGttctacgcaGAGggcgaccctaggctgag gaatggcgcctacgcctggtatggctttggctgtttctggTTTatcttcgcctactctatcgtgatgagcatcgtggccaagca gtatagccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttacctt ctccatgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacGATaactggacaGA GgtggcccactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcAGAaagacctatgatga ggagctgtttcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagc agcaatggcgagcgcctgcggagactgtct (SEQ ID NO:20) ChRmineAT:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactggtcttgctttatcgtgctgacaatcaccacattctacctgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttcttttccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctggcctgcccaatgctggtgtacgatctgctgtatcagctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctccatgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcc cactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID N0:21) ChRmineATS: atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactgggtgtgctttatcgtgctgacaatcaccacattctacctgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttcttttccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctggcctgcccaatgctggtgtacgatctgctgtatcagctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctcca tgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcc cactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID NO:22) ChRmineQTS:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactgggtgtgctttatcgtgctgacaatcaccacattctacctgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttctttgccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctggcctgcccaatgctggtgtacgatctgctgtataacctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccGAGttctacgcaGAGggcgaccctaggctgag gaatggcgcctacgcctggtatggctttggctgtttctggTTTatcttcgcctactctatcgtgatgagcatcgtggccaagca gtatagccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttacctt ctccatgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacGATaactggacaGA GgtggcccactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcAGAaagacctatgatga ggagctgtttcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagc agcaatggcgagcgcctgcggagactgtct (SEQ ID NO:23) ChRmineLTS:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactgggtgtgctttatcgtgctgacaatcaccacattctacgtgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttctttgccatgctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctggcctgcccaatgctggtgtacgatctgctgtatcagctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctcca tgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcc cactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID NO:24)12. ChRmineLTSA:Atggcacacgcaccaggcaccgaccagatgttctacgtgggcacaatggacggctggtatctggataccaagctgaact ccgtggccatcggcgcccactgggtgtgctttatcgtgctgacaatcaccacattctacgtgggctatgagtcctggacatctc ggggccctagcaagagaacctccttttacgccggctatcaggaggagcagaacctggccctgtttgtgaatttcttttccatg ctgagctacttcggcaagatcgtggccgacacactgggccacaacttcggcgatgtgggccccttcatcatcggcttcggc aattacaggtatgccgactacatgctggcctgcccaatgctggtgtacgatctgctgtatcagctgagggccccctatcgcgt gtcttgtagcgccatcatctttgccatcctgatgtctggcgtgctggccgagttctacgcagagggcgaccctaggctgagga atggcgcctacgcctggtatggctttggctgtttctggtttatcttcgcctactctatcgtgatgagcatcgtggccaagcagtata gccggctggcccagctggcccaggatacaggcgccgagcacagcctgcacgtgctgaagttcgccgtgtttaccttctcca tgctgtggattctgtttcccctggtgtgggccatctgccctagaggcttcggctggatcgacgataactggacagaggtggcc cactgcgtgtgcgacatcgtggccaagtcctgttacggctttgccctggcccggttcagaaagacctatgatgaggagctgtt tcggctgctggagcagctgggacacgacgaggatgagttccagaagctggagctggatatgaggctgagcagcaatgg cgagcgcctgcggagactgtct (SEQ ID NO:25)MethodsProtein expression and purification

[0174] Expression and purification of ChRmine from Rhodomonas lens was as previously described16. ChRmine was cloned into a custom vector with a human rhinovirus 3c cleavage site, linker sequence, superfolder GFP(sfGFP), and 7xHis tag for expression in insect cells. The construct was transformed into DHIOBac E. coli to generate a bacmid. Bacmid was transfected into adherent SF9 cells using Escort IV transfection reagent to produce P1 virus. SF9 cells (2 million cells / mL) were infected with P1 at a multiplicity of infection (MOI) ~0.1 to generate P2 virus. P2 virus was harvested 48-72 hours post infection. P3 virus was generated similarly. Sf9 cells (2-4 million cells / mL) were infected with P3 at a MOI ~2-5 for proteinoverexpression. 5 pM all-transretinal (ATR) was added to media 48 hours post infection. Cells were harvested 72 hours post infection by centrifugation (5000 * g, 15 min), frozen in liquid nitrogen, and stored at -80 °C.

[0175] Cells (-7.5-15 mL pellet) were thawed and resuspended in 100 mL of lysis buffer (50 mM Tris, 150 mM NaCI, 1 mM EDTA pH 8). Protease inhibitors were added immediately before use(1 pM E64, 1 pg / mL Pepstatin A, 10 pg / mL Soy Trypsin Inhibitor, 1 mM Benzimidine, 1 pg / mL Aprotinin, 1 pg / mL Leupeptin, 1 mM AEBSF, and 1 mMPMSF final concentrations). Benzonase (4 pl) was added after resuspension. Cells were lysed by sonication and were membranes pelleted by ultracentrifugation (150,000 x g, 45 minutes). The supernatant was discarded and membrane pellets were homogenized in extraction buffer (50 mM Tris, 150 mM NaCI, 1 mM EDTA, 1% n-Dodecyl-p-D-Maltopyranoside (DDM, Anatrace, Maumee, OH), pH 8). Homogenized membranes were stirred at 4 °C for 1-2 hr in 150 mL final volume. Insoluble material was pelleted at 33,000 x g for 45 minutes. Supernatant was bound to 5 mL of anti-GFP nanobody Sepharose resin for 1-2 hour at 4 °C. The resin was collected and washed with 10 mL buffer 1 (20 mM Tris, 150 mM NaCI, 1 mM EDTA, 0.025% DDM, pH 7.4), 40 mL buffer 2 (20 mM Tris, 150 mM NaCI, 0.025% DDM, pH 7.5), and 10 mL buffer 1. The resin was then resuspended in 6 mL of buffer 1 with 0.5 mg human rhinovirus 3c protease and rocked overnight at 4 °C. ChRmine was eluted with 8 mL buffer 1, spin concentrated to 500 pl with a 10 kDa cutoff Amicon Ultraspin concentrator (Millipore), and injected onto a Superose 6 increase column (GE Healthcare, Chicago, IL) on an NGC system (Bio-Rad, Hercules, CA) run in buffer 3 (20 mM Tris, 150 mM NaCI, 0.025% DDM, pH 7.4). Peak fractions containing ChRmine spin concentrated prior to incorporation into MSP1 E3D1 nanodiscs.

[0176] Purified ChRmine was reconstituted into MSP1 E3D1 nanodiscs and 2:1:1 molar ratio DOPE: POPS: POPC lipids at a final molar ratio of 1:4:400 (ChRmine:MSP1 E3D1 :lipid). 20 uM ChRmine, 80 uM MSP1 E3D1 , 8 mM lipid mix, and 5 mM DDM in buffer (20 mMTris pH 7.5, 150 mM NaCI) was used. Detergent was removed with 100 mg of washed Biobeads SM2 (BioRad) at 4 °C for -12 h. Sample was harvested, insoluble material was pelleted (21 ,000 x g, 10 min), and supernatant was injected onto a Superose 6 increase column run in 20 mM Tris, 150 mM NaCI, pH 7.5. Peak fractions corresponding to ChRmine protein in MSP1 E3D1 were collected for grid preparation.Cryo-EM sample preparation

[0177] ChRmine in nanosdiscs was concentrated (10 kDa cutoff) to -12 mg ml1and cleared by centrifugation at 21 ,000 g for 15 min at 4 °C prior to grid preparation. A 3-pl drop of protein was applied to freshly glow discharged Holey Carbon, 300 mesh R 1.2 / 1.3 gold grids(Quantifoil) . Samples were plunge frozen in liquid nitrogen cooled liquid ethane using a FEIVitrobot Mark IV (Thermo Fisher Scientific) at 4 °C, 100% humidity, 1 blot force, ~5 s wait time, and 3 s blot time. A 300 urn core fiber optic cable input was connected to a Lumencor light engine and its output was positioned in the Vitrobot chamber such that the plunging cryo-EM grid travels through an 8-10 mm diameter spot of light. LED light (peak wavelength 550 nm) was triggered at maximum power yielding a light intensity of 5-7.3 mW / mm2.Electrophysiology

[0178] ChRmine (residues 1-309) from Rhodomonas lens was cloned as a fusion to a Kir2.1 Golgi export sequence, the fluorophore mRuby2, and a Kv2.1 soma-targeting (ST) sequence (ChRmine-GE-mRuby2-ST). Mutagenesis was performed with inverse PCR. Constructs were transfected into cultured Chinese hamster ovary cells using Fugene or Lipofectamine transfection reagents. Patch clamp recording took place 1-2 days after transfection. Patch pipettes were pulled from borosilicate glass (Sutter Instruments) and filled with K-gluconate solution (in mM: 110 Kgluconate, 10 HEPES, 1 EGTA, 20 KCI, 2 MgCI2, 2 Na2ATP, 0.25 Na3GTP,10 Phosphocreatine, 295 mOsm, pH 7.45). Data was recorded at 1 kHz using 200b Multiclamp Axon Amplifier (Molecular Devices). Bath solution (in mM: 119 NaCI, 2.5 KCI, 1.3 MgSO4, 1.3 NaH2PO4, 20 glucose, 26 NaHCO3, 2.5 CaCI2) was maintained at 30-32° with inline heating. 5 pM all-trans retinal was added to bath solution prior to use.

[0179] Photostimulation was performed using a Spectra X light engine (Lumencor) with six color channels: violet, blue, cyan, teal, green, and red (center Wavelength / full-width at half maximum transmission 390 / 22 nm, 434 / 21 nm, 470 / 24 nm, 510 / 25 nm, 542 / 33 nm, and 648 / 20 nm, respectively).

[0180] Peak photocurrents are defined as the maximum current elicited by 5 ms teal light stimulation from a holding potential of -60 mV from successive sweeps with increasing light intensity. Sensitivity is defined as the light power to reach 50% peak photocurrent (5 ms 510 nm light stimulation, holding potential -60 mV) from a five-parameter logistic regression to photocurrent versus light intensity recorded from successive sweeps with varying LED power. Opening time constants are calculated from single exponential fits to current rise during 5 ms teal light stimulation at a holding potential of -60 mV. Closing time constants are calculated from single exponential fits to current decay after 5 ms teal light stimulation at a holding potential of - 60 mV. Steady state to peak current ratio is defined as the ratio of peak current and mean current during the final 20 ms elicited by a 500 ms teal light stimulus at a holding potential of -60 mV. Three successive sweeps with identical stimuli were averaged for kinetic measurements.References1. Zhang, F. et al. The Microbial Opsin Family of Optogenetic Tools. Cell 147, 1446-1457(2011).2. Deisseroth, K. & Hegemann, P. The form and function of channelrhodopsin. Science (NewYork, NY) 357, eaan5544-11 (2017).3. Schneider, F., Grimm, C. & Hegemann, P. Biophysics of Channelrhodopsin. Annu RevBiophys 44, 167-186 (2015).4. Kandori, H. Biophysics of rhodopsins and optogenetics. Biophysical Rev 12, 355-361 (2020).5. Stefanov, A. & Flannery, J. G. A Systematic Review of Optogenetic Vision Restoration:History, Challenges, and New Inventions from Bench to Bedside. Cold Spring Harb. Perspect. Med. 13, a041304 (2022).6. Lanzani, G. et al. Nanotechnology for vision restoration. Nat. Rev. Bioeng. 1-20 (2024) doi: 10.1038 / s44222-024-00210-4.7. Bi, A. et al. Ectopic Expression of a Microbial-Type Rhodopsin Restores Visual Responses inMice with Photoreceptor Degeneration. Neuron 50, 23-33 (2006).8. Gauvain, G. et al. Optogenetic therapy: high spatiotemporal resolution and pattern discrimination compatible with vision restoration in non-human primates. Commun. Biol. 4, 125 (2021).9. Chaffiol, A. et al. in vivo optogenetic stimulation of the primate retina activates the visual cortex after long-term transduction. Mol. Ther. - Methods Clin. Dev. 24, 1-10 (2022).10. Sahel, J. -A. et al. Partial recovery of visual function in a blind patient after optogenetic therapy. Nat Med 27, 1223-1229 (2021).11. Wright, W. et al. Restoring vision in mice with retinal degeneration using multicharacteristic opsin. Neurophotonics 4, 041505-041505 (2017).12. Batabyal, S. et al. Sensitization of ON-bipolar cells with ambient light activatable multicharacteristic opsin rescues vision in mice. Gene Ther. 28, 162-176 (2021).13. Lu, Q., Ganjawala, T. H., Krstevski, A., Abrams, G. W. & Pan, Z.-H. Comparison of AAV-Mediated Optogenetic Vision Restoration between Retinal Ganglion Cell Expression and ON Bipolar Cell Targeting. Mol. Ther. - Methods Clin. Dev. 18, 15-23 (2020).14. Ganjawala, T. H., Lu, Q., Fenner, M. D., Abrams, G. W. & Pan, Z.-H. Improved CoChRVariants Restore Visual Acuity and Contrast Sensitivity in a Mouse Model of Blindness under Ambient Light Conditions. Mol. Ther. 27, 1195-1205 (2019).15. Bansal, H., Pyari, G. & Roy, S. Theoretical prediction of broadband ambient light optogenetic vision restoration with ChRmine and its mutants. Sci. Rep. 14, 11642 (2024).16. Tucker, K., Sridharan, S., Adesnik, H. & Brohawn, S. G. Cryo-EM structures of the channelrhodopsin ChRmine in lipid nanodiscs. Nat Commun 13, 4842 (2022).17. Kishi, K. E. et al. Structural basis for channel conduction in the pump-like channelrhodopsinChRmine. Cell (2022) doi:10.1016 / j.cell.2022.01.007.18. Sridharan, S. et al. High-performance microbial opsins for spatially and temporally precise perturbations of large neuronal networks. Neuron (2022) doi:10.1016 / j. neuron.2022.01.008.19. Marshel, J. H. et al. Cortical layer-specific critical dynamics triggering perception. Science365, eaaw5202 (2019).20. Zerche, M. et al. Efficient and sustained optogenetic control of nervous and cardiac systems. bioRxiv 2023.11.17.567544 (2023) doi: 10.1101 / 2023.11.17.567544.

Claims

CLAIMS1. A nucleic acid comprising a nucleotide sequence encoding a channelrhodopsin variant comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 and comprising one or more of the following substitutions relative to SEQ ID NO:1 : i. an amino acid substitution of the alanine at position 81 (A81); ii. an amino acid substitution of the leucine at position 47 (L47); iii. an amino acid substitution of the glutamine at position 130 (Q130); iv. an amino acid substitution of the threonine at position 119 (T119); and v. an amino acid substitution of the serine at position 35 (S35).

2. The nucleic acid of claim 1 , wherein the amino acid substitution at A81 is A81S, A81T, A81 N, or A81Q.

3. The nucleic acid of claim 1 or 2, wherein the amino acid substitution at L47 is L47V, L47A, L47I, L47M, L47F, L47Y, or L47W.

4. The nucleic acid of any one of claims 1-3, wherein the amino acid substitution at Q130 is Q130N, Q130S, or Q130T.

5. The nucleic acid of any one of claims 1-4, wherein the amino acid substitution at T119 is T119A, T119V, T119I, T119L, T119M, T119F, T119Y, or T119W.

6. The nucleic acid of any one of claims 1-5, wherein the amino acid substitution at S35 is S35V, S35A, S35I, S35L, S35M, S35F, S35Y, or S35W.

7. The nucleic acid of any one of claims 1-6, comprising one of the following double substitutions, triple substitutions, or quadruple substitution:L47V T119A;Q130N T119A;A81S T119A;A81S T119A S35V;Q130N T119A S35V;L47V T119A S35V; andL47V T119A S35V A81S.

8. The nucleic acid of any one of claims 1-7, wherein the amino acid sequence has at least 85%, at least 90%, at least 92%, at least 95% at least 96% at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:1.

9. The nucleic acid of claim 1 , wherein the channelrhodopsin variant comprises the amino acid sequence set forth in any one of SEQ ID NOs:2-13.

10. The nucleic acid of any one of claims 1-9, wherein the nucleotide sequence has at least 85% identity to the nucleotide sequence of one of:SEQ ID NOs:14-25.

11. The nucleic acid of any one of claims 1-10, wherein(i) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO: 14 and the channelrhodopsin variant comprises the amino acid substitution A81S,(ii) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO: 15 and the channelrhodopsin variant comprises the amino acid substitution L47V,(iii) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO: 16 and the channelrhodopsin variant comprises the amino acid substitution Q130N,(iv) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO: 17 and the channelrhodopsin variant comprises the amino acid substitution S35V,(v) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO: 18 and the channelrhodopsin variant comprises the amino acid substitution T119A,(vi) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO: 19 and the channelrhodopsin variant comprises the amino acid substitutions L47V T119A,(vii) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO:20 and the channelrhodopsin variant comprises the amino acid substitutions Q130N T119A,(viii) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO:21 and the channelrhodopsin variant comprises the amino acid substitutions A81 S T119A,(ix) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO:22 and the channelrhodopsin variant comprises the amino acid substitution A81 S T119A S35V,(x) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO:23 and the channelrhodopsin variant comprises the amino acid substitutions Q130N T119A S35V,(xi) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO:24 and the channelrhodopsin variant comprises the amino acid substitutions L47V T119A S35V, or(xii) the nucleotide sequence has at least 85% identity to the nucleotide sequence of SEQ ID NO:25 and the channelrhodopsin variant comprises the amino acid substitutions L47V T119A S35V A81S.

12. The nucleic acid of any one of claims 1-11 , comprising a nucleotide sequence encoding a Golgi export signal, wherein the nucleotide sequence encoding the channelrhodopsin variant is a first nucleotide sequence and the nucleotide sequence encoding the Golgi export signal is a second nucleotide sequence, wherein the first nucleotide sequence is upstream of and in frame with the second nucleotide sequence and the channelrhodopsin variant is fused at the C-terminus to the Golgi export signal.

13. The nucleic acid of claim 12, further comprising a third nucleotide sequence encoding a SOMA targeting motif, wherein the third nucleotide sequence is downstream to and in frame with the second nucleotide sequence and the SOMA targeting motif is fused to the C-terminus of the Golgi export signal.

14. A vector comprising the nucleic acid of any one of claims 1-13 operatively linked to an expression control element.

15. The vector of claim 14, wherein the vector is a viral vector.

16. The vector of claim 15, wherein the viral vector is an adenoviral vector or a lentiviral vector.

17. The vector of any one of claims 14-16, wherein the expression control element comprises a promoter selected from the group consisting of a SNCG promoter, a CAG promoter, a mini CAG promoter, a CBh promoter, a NEFH promoter, a GRK1 promoter, a RLBP1 promoter, a VMD2 promoter, a Syn1 promoter and a Syn1 (enhSynl) promoter.

18. A cell comprising the nucleic acid of any one of claims 1-13 or the vector of any one of claims 14-17.

19. The cell of claim 18, wherein the cell is a mammalian cell.

20. The cell of claim 19, wherein the mammalian cell is a retinal cell or cardiac cell.

21. The cell of claim 19 or 20, wherein the mammalian cell is a neuron.

22. A transgenic mammal comprising the cell of any one of claims 19-21.

23. The cell of any one of claims 18-22, wherein the channelrhodopsin variant is post- translationally modified by removal of the N-terminal methionine and conversion of the alanine at position 2 to acetyl-alanine.

24. A method of expressing a channelrhodopsin variant in a mammalian subject, the method comprising delivering the nucleic acid of any one of claims 1-13 or the vector of any one of claims 14-17 or the cell of any one of claims 19-21 to the eye of the subject.

25. The method of claim 24, wherein the subject has age-related macular degeneration or retinitis pigmentosa.

26. The method of claim 24 or 25, wherein the method comprises administering a therapeutically effective amount of the nucleic acid, the vector, or the cell to the retina of the subject.

27. The method of claim 26, wherein the administering comprises an intraocular injection or an intraocular infusion.

28. The method of claim 27, wherein the intraocular injection comprises intravitreal injection, subretinal injection, or suprachoroidal injection.

29. The method of claim 27, wherein the intraocular infusion is an intravitreal infusion, a subretinal infusion, or a suprachoroidal infusion.

30. A channelrhodopsin variant comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 and comprising one or more of the following substitutions relative to SEQ ID NO:1 : i. an amino acid substitution of the alanine at amino acid 81 (A81); ii. an amino acid substitution of the leucine at position 47 (L47); iii. an amino acid substitution of the glutamine at position 130 (Q130); iv. an amino acid substitution of the threonine at position 119 (T119); and v. an amino acid substitution of the serine at position 35 (S35).

31. The channelrhodopsin variant of claim 30, wherein the amino acid substitution at A81 is A81S, A81T, A81 N, or A81Q.

32. The channelrhodopsin variant of claim 30 or 31 , wherein the amino acid substitution at L47 is L47V, L47A, L47I, L47M, L47F, L47Y, or L47W.

33. The channelrhodopsin variant of any one of claims 30-32, wherein the amino acid substitution at Q130 is Q130N, Q130S, or Q130T.

34. The channelrhodopsin variant of any one of claims 30-33, wherein the amino acid substitution at T119 is T119A, T119V, T119I, T119L, T119M, T119F, T119Y, or T119W.

35. The channelrhodopsin variant of any one of claims 30-34, wherein the amino acid substitution at S35 is S35V, S35A, S35I, S35L, S35M, S35F, S35Y, or S35W.

36. The channelrhodopsin variant of any one of claims 30-35, comprising one of the following double substitutions, triple substitutions, or quadruple substitution:L47V T119A;Q130N T119A;A81S T119A;A81S T119A S35V;Q130N T119A S35V;L47 T119A S35V; and L47V T119A S35V A81S.

37. The channelrhodopsin variant of any one of claims 30-36, wherein the amino acid sequence has at least 85%, at least 90%, at least 92%, at least 95% at least 96% at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:1.

38. The channelrhodopsin variant of claim 30, comprising the amino acid sequence set forth in any one of SEQ ID NOs:26-37.

39. A transgenic mammal expressing the channelrhodopsin variant of any one of claims SO- 38.

40. A retinal prosthetic device comprising a retinal ganglion cell, the cell expressing the channelrhodopsin variant of any one of claims 30-39.

41. A method of determining an effect of an agent on a property of the channelrhodopsin variant of any one of claims 30-39, the method comprising: exposing the channelrhodopsin variant to light in the presence of and in the absence of the agent; and comparing one or more of the following properties of the channelrhodopsin variant: photocurrent, sensitivity, closing speed, opening speed, and desensitization in the presence of and in the absence of the agent, wherein presence of a difference in one or more the properties in presence of the agent identifies the agent as having an effect on the property.

42. The method of claim 41, wherein the agent is a small molecule.

43. The method of claim 41 or 42, wherein the channelrhodopsin variant is present in lipid nanodiscs or in cells.

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