Fig. 1 Schematic diagram for the optogenetic tools responsive to ultraviolet and violet light(a) In the presence of UV light, homodimerized UVR8 dissociates into monomers and its fused proteins also depolymerizes. (b) Homodimerized UVR8 cannot bind to its ligand COP1 under dark conditions, but UVR8 binds to COP1 to form a heterodimer upon UV light illumination. (c) Violet light induces irreversible self-photocleavage of PhoC1 resulting in the quenching of green fluorescence.POI—protein of interest

Fig. 2 Schematic diagram for the optogenetic tools responsive to blue light(a) The blue light-responsive cation channel protein ChR2 is activated to induce influx of Ca2+ and Na+ by blue light illumination. (b) Under blue light illumination conditions, the chromophore retinal is isomerized to lead to the conformational changes of melanopsin thereby activating phospholipase C (PLC) through G protein (Gαq) and phospholipase C (PLC), which triggers Ca2+ influx by activating transient receptor potential ion channels (TRPCs) on the cell membrane and from the endoplasmic reticulum (ER). (c) With blue light illumination, a light-induced conformation is developed between the AsLOV protein core and the flavoprotein FMN, which results in undocking and unwinding of the LOV2 C-terminal Jα helix. (d) With blue light illumination, a light-induced conformation is formed between the cpLOV and the flavoprotein FMN, which leads to undocking and unwinding of the LOV2 C-terminal Jα helix. (e) Blue light induces the homodimerization of VVD, thus enabling proximity of the fused proteins. (f) Blue light induces the heterodimerization of pMag and nMag, thus enabling proximity of the fused target protein (g) LOVTRAP is a blue light responsive protein dissociation system, which is a reversible light-induced protein system. Zdk1 binds to LOV domain to form a heterodimer under dark conditions, but dissociates from LOV domain upon blue light illumination. (h) Under dark conditions, the N-terminal LOV photoreceptive domain of the light-sensitive protein EL222 binds to its C-terminal HTH DNA-binding domain, thus preventing EL222 dimerization and DNA binding, while blue light irradiation enables EL222 dimerization to recognize its target DNA sequences. (i) Blue light triggers heterodimerization of CRY2 and CIB1 to initiate gene expression. (j) Blue light triggers oligomerization of CRY2, while CRY2 forms a monomer under dark conditions. (k) Blue light triggers multimerization of CRY2clust/olig.

Fig. 3 Schematic diagram for the optogenetic tools responsive to cyan and yellow light(a) The fluorescent proteins Dronpa145N and Dronpa145K dimerizes under dark conditions, while the Dronpa protein undergoes a conformational change that enables depolymerization into monomers in the presence of cyan light. (b) Under dark conditions, pdDronpa homodimerizes and thus blocks the active sites of target proteins, while blue light illumination causes dissociation of the homodimers to expose the active sites of target proteins. (c) Cyan light illumination causes dissociation of the homotetramerized TtCBD. (d) Synthetic light-gated GPCRs (Opto-XRs) undergoes a conformational change that enables the activation of G-protein-mediated intracellular signaling cascades by cyan light illumination. (e) Yellow light-mediated activation of NpHR which triggers Cl- infux.

Fig. 4 Schematic diagram for the optogenetic tools responsive to red/far red light(a) Phytochrome B (PhyB) maintains at Pr form which is biologically inactive in the dark. Red light illumination converts PhyB into the Pfr form and induces heterodimerization with PIF6 in the presence of the photosensitive pigment PCB. (b) In the dark, truncated Phytochrome A (ΔPhyA) maintains at Pr form, red light illumination converts ΔPhyA into the Pfr form and induces heterodimerization with FHY1 in the presence of the photosensitive pigment PCB. (c) Red light induces dissociation of homodimerized BphP1 which can interact with PpsR2 to form a heterodimerization pair.

Fig. 5 Development and application of optogenetic tools in neurobiology(a) Three major types of opsins used in neurobiology. The blue light-responsive cation channel protein ChR2 is activated to induce influx of Ca2+ and Na+ and trigger an action potential. The yellow light-responsive NpHR is activated to allow chloride ions to enter the cytoplasm. Synthetic light-gated GPCRs (Opto-XRs) is engineered by replacing the intracellular domain of rhodopsin with that of G protein-coupled receptors to enable G-protein mediated intracellular signaling cascades. (b) Genetic targeting of ChR2 into the amygdala region of mouse brain enables optical control of the neural pathway of the mouse-hunting behavior by implanting optical fibers. (c) Genetic targeting of ChR2/NpHR into the cervical dorsal spinal cord of mice enables specific activation or suppression of the neurons, and avoids stimulation of non-targeted cells. (d) ChR2 is delivered into the deep brain neurons of mice by coupling with lanthanide-doped upconversion nanoparticles (UCNP), which can convert blue light to tissue-penetrable NIR light to activate ChR2 expressed dopaminergic neurons.

Fig. 6 Optogenetic tools used for tumor therapy(a) Photochemokine receptors used for immunotherapy in murine melanoma. Under 505 nm light illumination, the chimeric photoactivated chemokine receptor composed of rhodopsin α subunit and chemokine receptor-4 (CXCR4) is activated to induce T cell polarization, resulting in the inhibition of tumor growth. (b) Opto-CRAC for cellular immunotherapy. Under blue light illumination, the Jα helix at the carboxyl terminus of LOV2 domain dislocates to expose the C-terminus of STIM1 protein, which stimulates the ORAI1 Ca2+ channel to initiate the calcium-dependent cascade and

Fig. 7 Applications of optogenetic tools in treating cardiovascular diseases(a) Cyanobacterial photosynthetic systems for myocardial ischemia treatment. The cyanobacteria are injected into the hearts of the acute myocardial infarction model rats to produce oxygen through photosynthesis under light illumination conditions, which increases the metabolic activity of cardiomyocytes to improve ventricular function and alleviate acute tissue ischemia. (b) Optogenetic pacemakers. The non-selective cation channel ChR2 is used to control the cardiac excitability. (c) Inhibitory photosensitive protein systems for cardiovascular diseases treatment. ARCH-T mediated H+ efflux can inhibit myocardial activity and alleviate arrhythmia under yellow light illumination. (d) Optogenetic pacemakers for modulating cardiomyocyte activity. Melanopsin, a photoactivated G-protein-coupled receptor, activates the phospholipase C and catalyzes the hydrolysis of PIP2 to produce IP3, enabling release of Ca2+ and enhancement of the pacing activity of cardiomyocytes under blue light (470 nm) illumination conditions.

Fig. 8 Optogenetic tools for diabetes therapy(a) Melanopsin-based blue light regulatory systems for diabetes treatment. Upon blue light stimulation, the melanopsin conformation is changed, thereby activating phospholipase C (PLC) through G protein (Gαq) and phospholipase C (PLC), which triggers Ca2+ influx by the activation of transient receptor potential ion channels (TRPCs) on the cell membrane and from the endoplasmic reticulum (ER). The activated NFAT translocates into the nucleus, and binds to its specific promoter (PNFAT), which initiates the GLP-1 gene expression to control blood glucose homeostasis. (b) The light-oxygen-voltage domain-based blue light regulatory system for diabetes treatment. In the presence of substrate, blue light is produced by the luciferase-catalyzed reaction, leading to the dimerization of the photosensitive protein Vivid. The DNA binding domain Gal4 (1-65 aa) fuses with Vivid and VP16 for incorporation into the nucleus to bind to the DNA operator (5 × UAS), which initiates the expression of insulin to control blood glucose homeostasis. (c) ΔPhyA-based red light regulatory systems for diabetes treatment. Under red light illumination, the hybrid transactivator FHY1-VP64 can be translocated into nucleus by photosensitive DNA binding elements (ΔPhyA-Gal4), in which it can bind to a particular operon sequence (5×UAS) to initiate the expression of insulin. The microcapsules containing engineered cells are implanted into the back of diabetic mice, through which the cells can be induced to produce insulin to control blood glucose homeostasis under red light illumination conditions.

Fig. 9 Light-controlled DNA recombination based on split-Cre recombinase(a) Photoactivatable Cre recombinase systems based on Cry2-CIBN. Under dark conditions, Cre recombinase is dissociated into two inactive parts. Under blue light illumination, CRY2 undergoes a conformational change and binds to CIBN, which induces CreC and CreN to form a complete Cre recombinase for restoration of its activity. (b) Photo-controlled Cre recombinase systems based on Magnet (pMag and nMag). Blue light illumination enables the dimerization of pMag and nMag to reconstitute a complete Cre recombinase to permit light-dependent DNA recombination. (c) A far-red light-inducible split Cre-loxP (FISC) system. Under far-red light illumination, the photosensitive protein BphS converts GTP into c-di-GMP within cells, which triggers the dimerization of BldD to fuse with transcriptional activator p65-VP64 for translocation into the nucleus to initiate the expression of DocS-CreC. Constitutive expressed CreN-Coh2 is driven by the CMV promoter. The catalytic activities of Cre recombinase can be restored through affinity interactions of their respective Coh2 and DocS fusion domains.

Fig. 10 Light-controlled gene editing systems based on CRISPR-Cas9(a) Photoactivatable CRISPR-Cas9 systems based on pMag-nMag. Under dark conditions, Cas9 is splitted into two fragments without nuclease activity. With blue light illumination, the NCas9 and CCas9 domains can be reassociated to form a complete Cas9 by the light-dependent dimerization of pMag and nMag, thereby reconstituting editing for targeted genes. (b) Photoactivatable CRISPR-Cas9 systems based on protected sgRNA. Under dark conditions, the seed sequence of sgRNA is bound by an oligonucleotide that could be cleaved by UV light. With UV light illumination, the oligonucleotide is broken, and the seed sequence of sgRNA is exposed to allow Cas9 to bind to and cleave the target DNA. (c) Near-infrared light-regulated CRISPR-Cas9 systems based on UCNPs. The Cas9-sgRNA complex is wrapped on the outside of UCNPs by PEI and SiO2, which have the capability to convert near-infrared light (980 nm) to ultraviolet light for the Cas9-sgRNA complex to bind to target genes. (d) CRISPR-Cas9 systems based on APC gold nanoparticles. Cas9 plasmids carrying the heat-induced promoter can be efficiently delivered into mice by APC gold nanoparticles. Under near infrared light irradiation at 1064 nm, APC gold nanoparticles can convert light energy to heat, which activates the expression of Cas9 nuclease for genome editing. (e) A far-red light-activated split-Cas9 (FAST) system. The far-red light activates the expression of the fusion proteinNCas9-Coh2, and the CMV promoter drives the expression of the fusion protein DocS-CCas9, which consequently activates Cas9 nuclease by heterodimerization between Coh2 and DocS.

Fig. 11 Light-controlled gene transcription systems based on CRISPR-dCas9(a) Photoactivable dCas9-meidated transcription systems based on CRY2-CIBN. Under dark conditions, dCas9 is splitted into two fragments without catalytic activity. With blue-light illumination, CRY2 undergoes a conformational change that enables interactions with CIBN, which causes translocation of the transactivator VP64 to activate downstream gene transcription. (b) Blue light-controlled dCas9-meidated gene transcription systems based on SAM and pMag-nMag. SAM, a synergistic activation mediator that extends guide RNAs with an insertion of a MS2-box sequence into the loop of gRNA, can recruit effector protein to initiate gene transcription. Under blue-light illumination conditions, the NdCas9 and CdCas9 domains can be reassociated to form a complete dCas9 by the light-dependent dimerization of pMag and nMag, thereby activating the downstream gene transcription. (c) A far red light-controlled gene transcription system based on SAM and BphS-BldD. Under far-red light illumination conditions, the fused protein MS2-p65-HSF1 can express to activate the target gene transcription. (d) Light-controlled dCas9-midated gene transcription systems based on REDMAP. Under red-light illumination conditions, the heterodimerization of ΔPhyA and FHY1 enables the expression of the fused protein MS2-p65-HSF1 to activate endogenous gene expression by sgRNAs-mediated recruitment of the transcriptional activator domain.

Fig. 12 Light-controlled gene transcription and editing systems based on CRISPR-Cas12a/dCas12a(a) Blue light-controlled split-Cas12a gene editing systems. Under blue-light illumination conditions, pMag and nMagHigh1 that are fused to dCas12a are reassociated by the dimerization of pMag and nMag, thereby recovering the catalytic activity of Cas12a to cleave the target DNA sequence. (b) Far-red light-controlled Cas12a gene editing systems. Under far-red light illumination conditions, BphS can convert GTP into c-di-GMP to trigger the dimerization of p65-HSF1-BldD for binding with the operator to induce the expression of Cas12a for targeted genome cleavage. (c) Far-red light-controlled gene transcription systems based on SunTag and BphS-BldD. Under far-red light illumination conditions, the expression of the fusion protein dCas12a-GCN4 can be induced to activate the target gene transcription by the recruitment of the transactivator fused with ScFv.

Fig. 13 Light-controlled transgene expression systems based on RNA level(a) Light-controlled transgene expression systems based on PAL. Under dark conditions, PAL is dissociated from the RNA aptamer to initiate reporter gene expression. With blue light illumination, PAL can bind to RNA aptamer, which can suppress the reporter gene expression. (b) Light-controlled transgene expression systems based on LicV. A stem-loop inserted termination sequence hinders the reporter gene expression, and under blue light illumination, the dimerized VVDs enables dimerization of CATs, which binds to the RAT sequence and unfold the stem-loop, thereby restoring the reporter gene expression.

Fig. 14 Light-controlled movement and localization of organelles(a) Light-controlled organelle localization based on CRY2-CIB1. Under blue-light illumination conditions, CRY2 undergoes a conformational change that enables interactions with CIBN for the movement of organelles driven by the molecular motor. (b) Light-controlled localization of organelles based on LOV. Under blue light irradiation conditions, the organelles fused with PEX-LOV can bind to the engineered PDZ domain ePDZb1fused with molecular motor, leading to the movement and localization of organelle.

Fig. 15 Optogenetic tools for intelligent bioelectronic medicine(a) A brain-controlled wireless-powered optogenetic implant device for transgene expression. This brain-controlled transgenic expression device can wirelessly control gene expression through human brain activities. Electroencephalogram (EEG) headset captures brain wave activities and transmits them to the field intensity generator interface (BCI) through Bluetooth, which integrates with a near-infrared LED. The bacterial diguanylate cyclase (DGC) can be activated by near-infrared light, which converts GTP into c-di-GMP for the activation of the STING signal pathway to initiate the transgene expression. (b) Semi-automatic intelligent diagnosis and treatment systems based on optogenetic designer cells for diabetes treatment. The blood glucose value detected from the blood glucose monitor can be automatically transmitted to the smart controller and smart phone through Bluetooth. The smart controller can regulate far-red light intensity based on the blood glucose value. Under far-red light irradiation, BphS converts intracellular GTP into c-di-GMP, which dimerizes the hybrid transcriptional activator p65-VP64-BldD into the nucleus for binding to the chimeric promoter to initiate the expression of insulin or GLP-1 for controlling blood glucose homeostasis. (c) Wearable smart watch-controlled optogenetic systems for disbetes treatment. The system utilizes green light from a smartwatch to activate artificially customized cells implanted into the skin of mice. The green light-responsive TtCBD is anchored onto the cell membrane. When the LEDs are turned on, TtCBD is depolymerized, and the hybrid transcriptional activator TetR-VPR is separated from the cell membrane to initiate the transcription expression of GLP-1for controlling blood glucose homeostasis.