Optogenetics for transcriptional programming and genetic engineering

Naturally evolved or engineered photosensory modules have been harnessed for transcriptional control and genetic engineering in space and time by using photons emitting in the wide range of 200–1000 nm.Optogenetic modules can be modularly engineered into host cells to control physiological processes via light-switchable allosteric control, oligomeric transition, protein–protein heterodimerization, and self-cleavage.Optogenetic devices can be integrated with the transcriptional machinery, DNA recombinases, RNA-binding proteins, and CRISPR-based genome-engineering tools to precisely manipulate transcriptional reprogramming, DNA/RNA modifications, genetic recombination, and genome/epigenome editing.Optogenetics can be combined with synthetic biology, biophotonics, and genome-engineering approaches to advance precision medicine and personalized therapies for human diseases. Optogenetics combines genetics and biophotonics to enable noninvasive control of biological processes with high spatiotemporal precision. When engineered into protein machineries that govern the cellular information flow as depicted in the central dogma, multiple genetically encoded non-opsin photosensory modules have been harnessed to modulate gene transcription, DNA or RNA modifications, DNA recombination, and genome engineering by utilizing photons emitting in the wide range of 200–1000 nm. We present herein generally applicable modular strategies for optogenetic engineering and highlight latest advances in the broad applications of opsin-free optogenetics to program transcriptional outputs and precisely manipulate the mammalian genome, epigenome, and epitranscriptome. We also discuss current challenges and future trends in opsin-free optogenetics, which has been rapidly evolving to meet the growing needs in synthetic biology and genetics research. Optogenetics combines genetics and biophotonics to enable noninvasive control of biological processes with high spatiotemporal precision. When engineered into protein machineries that govern the cellular information flow as depicted in the central dogma, multiple genetically encoded non-opsin photosensory modules have been harnessed to modulate gene transcription, DNA or RNA modifications, DNA recombination, and genome engineering by utilizing photons emitting in the wide range of 200–1000 nm. We present herein generally applicable modular strategies for optogenetic engineering and highlight latest advances in the broad applications of opsin-free optogenetics to program transcriptional outputs and precisely manipulate the mammalian genome, epigenome, and epitranscriptome. We also discuss current challenges and future trends in opsin-free optogenetics, which has been rapidly evolving to meet the growing needs in synthetic biology and genetics research. Plants and microbes have evolved a plethora of photoreceptors capable of harnessing photons to modulate gene expression in response to environmental cues, thereby benefiting their own life cycles [1.Tan P. et al.Optophysiology: illuminating cell physiology with optogenetics.Physiol. Rev. 2022; 102: 1263-1325Crossref PubMed Scopus (0) Google Scholar]. Upon exposure to light emitting in the range of 200–800 nm, light-absorbing cofactors or chromophores within photoreceptors undergo photochemical reactions to trigger conformational changes, which in turn allosterically regulate adjacent domains and/or alter their interactions with cognate binding partners via homomultimerization, heterodimerization, or dissociation [2.Dagliyan O. Hahn K.M. Controlling protein conformation with light.Curr. Opin. Struct. Biol. 2019; 57: 17-22Crossref PubMed Scopus (16) Google Scholar, 3.Lu X. et al.Engineering photosensory modules of non-opsin-based optogenetic actuators.Int. J. Mol. Sci. 2020; 21: 6522Crossref Scopus (8) Google Scholar, 4.Seong J. Lin M.Z. Optobiochemistry: genetically encoded control of protein activity by light.Annu. Rev. Biochem. 2021; 90: 475-501Crossref PubMed Scopus (9) Google Scholar, 5.Losi A. et al.Blue-light receptors for optogenetics.Chem. Rev. 2018; 118: 10659-10709Crossref PubMed Scopus (115) Google Scholar, 6.Zhang K. Cui B. Optogenetic control of intracellular signaling pathways.Trends Biotechnol. 2015; 33: 92-100Abstract Full Text Full Text PDF PubMed Google Scholar, 7.Toettcher J.E. et al.The promise of optogenetics in cell biology: interrogating molecular circuits in space and time.Nat. Methods. 2011; 8: 35-38Crossref PubMed Scopus (181) Google Scholar, 8.Tan P. et al.Optogenetic immunomodulation: shedding light on antitumor immunity.Trends Biotechnol. 2017; 35: 215-226Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar] (Figure 1). Following light withdrawal, the photoreceptors readily return to their ground states to enable reversible control over the involved biological processes. Over the past 5 years, these non-mammalian photosensitive modules (PSMs) have been successfully engineered into the transcription machinery, DNA recombinases, and genome or epigenome (see Glossary) editing tools [9.Wang D. et al.CRISPR-based therapeutic genome editing: strategies and in vivo delivery by AAV vectors.Cell. 2020; 181: 136-150Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 10.Nakamura M. et al.CRISPR technologies for precise epigenome editing.Nat. Cell Biol. 2021; 23: 11-22Crossref PubMed Scopus (67) Google Scholar, 11.Doudna J.A. The promise and challenge of therapeutic genome editing.Nature. 2020; 578: 229-236Crossref PubMed Scopus (302) Google Scholar], thereby enabling precise control over key molecular steps involved in the regulation of the ‘central dogma’ of genetics (Table 1, Key table).Table 1Key table. Optogenetic devices commonly used for transcriptional programming and genetic studiesToolsCofactor or chromophoreWavelength(s)Key attributesSelected examplesI. Allosteric controlLOV2 or cpLOV2FMN450–470 nm (on)Dark (off)Modular and highly transferrable across protein scaffolds or templates;rapid reversibility with on/off half-lives in seconds.Opto-CRAC and CaRROT for Ca2+-dependent transcriptional activation [14.He L. et al.Near-infrared photoactivatable control of Ca(2+) signaling and optogenetic immunomodulation.Elife. 2015; 4e10024Crossref Google Scholar,83.Nguyen N.T. et al.Rewiring calcium signaling for precise transcriptional reprogramming.ACS Synth. Biol. 2018; 7: 814-821Crossref PubMed Scopus (32) Google Scholar, 84.Nguyen N.T. et al.Optogenetic approaches to control Ca(2+)-modulated physiological processes.Curr. Opin. Physiol. 2020; 17: 187-196Crossref PubMed Scopus (6) Google Scholar, 85.Nguyen N.T. et al.CRAC channel-based optogenetics.Cell Calcium. 2018; 75: 79-88Crossref PubMed Scopus (17) Google Scholar, 86.Ma G. et al.Optogenetic toolkit for precise control of calcium signaling.Cell Calcium. 2017; 64: 36-46Crossref PubMed Scopus (2) Google Scholar];LiCre for photoactivatable DNA recombination [59.Hughes R.M. et al.Light-mediated control of DNA transcription in yeast.Methods. 2012; 58: 385-391Crossref PubMed Scopus (64) Google Scholar];LiCASINO [93.Polstein L.R. Gersbach C.A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.Nat. Chem. Biol. 2015; 11: 198-200Crossref PubMed Scopus (431) Google Scholar] or CASANOVA [97.Wang X. et al.A far-red light-inducible CRISPR-Cas12a platform for remote-controlled genome editing and gene activation.Sci. Adv. 2021; 7eabh2358Crossref Scopus (2) Google Scholar] for photoswitchable Cas9 inhibition;paCBE for light-inducible cytosine base editing [93.Polstein L.R. Gersbach C.A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.Nat. Chem. Biol. 2015; 11: 198-200Crossref PubMed Scopus (431) Google Scholar].PYPp-Coumaric acid450 nm (on)Dark (off)Undergoes robust conformational changes;tailored for both on/off photoswitches with half-lives within milliseconds.opto-DN-CREB for CREB-dependent gene regulation [31.Ali A.M. et al.Optogenetic inhibitor of the transcription factor CREB.Chem. Biol. 2015; 22: 1531-1539Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar].II. Oligomeric switchCRY2FAD~470 nm (oligomer)Dark (monomer)Tunable oligomeric states with CRY2olig and CRY2clust;reversible with on/off half-lives in seconds and minutes.mRNA-LARIAT for mRNA perturbation and relocalization [111.Statello L. et al.Gene regulation by long non-coding RNAs and its biological functions.Nat. Rev. Mol. Cell Biol. 2021; 22: 96-118Crossref PubMed Scopus (911) Google Scholar].EL222FMN~450 nm (dimer)Dark (monomer)Light-induced dimerization for DNA binding.VP-EL222 for gene expression [43.Motta-Mena L.B. et al.An optogenetic gene expression system with rapid activation and deactivation kinetics.Nat. Chem. Biol. 2014; 10: 196-202Crossref PubMed Scopus (235) Google Scholar].RsLOVFMN~450 nm (monomer)Dark (dimer)Monomerizes upon blue light stimulation.Cas9-RsLOV for Cas9 activity tuning [45.Richter F. et al.Engineering of temperature- and light-switchable Cas9 variants.Nucleic Acids Res. 2016; 44: 10003-10014PubMed Google Scholar].AuLOVFMN~450 nm (dimer)Dark (monomer)Exists as monomer in the dark and dimerizes upon blue light stimulation.Opto-RTKs to trigger downstream effectors (Ras/ERK and PI3K/AKT) and modulate gene expression [47.Grusch M. et al.Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.EMBO J. 2014; 33: 1713-1726Crossref PubMed Scopus (173) Google Scholar,48.Khamo J.S. et al.Optogenetic delineation of receptor tyrosine kinase subcircuits in PC12 cell differentiation.Cell Chem. Biol. 2019; 26: 400-410Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar].pdDronpa1CYG triad400 nm (dimer)500 nm (monomer)Dimerizes in the dark and dissociates into monomers by cyan light.ps-Cas9 or ps-dCas9 for (epi)genome editing and transcriptional programming [52.Zhou X.X. et al.Optical control of cell signaling by single-chain photoswitchable kinases.Science. 2017; 355: 836-842Crossref PubMed Scopus (106) Google Scholar].III. Protein–protein heterodimerizationiLID(LOV2-ssrA + sspB)FMN~470 nm (on)Dark (off)Blue light-responsive iLID variants with nM to mM affinities.CasDrop and Corelet for phase separation and transcriptional regulation [119.Wang T. et al.Red-shifted optogenetics comes to the spotlight.Clin. Transl. Med. 2022; 2022e807Google Scholar,120.Shin Y. et al.Liquid nuclear condensates mechanically sense and restructure the genome.Cell. 2018; 175: 1481-1491Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar]; OptoCAR or LiCAR T cells for photoswitchable cellular immunotherapy [15.He L. et al.Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering.Nat. Chem. Biol. 2021; 17: 915-923Crossref PubMed Scopus (7) Google Scholar,18.Nguyen N.T. et al.Nano-optogenetic engineering of CAR T cells for precision immunotherapy with enhanced safety.Nat. Nanotechnol. 2021; 16: 1424-1434Crossref PubMed Scopus (5) Google Scholar].Magnet (pMag/nMag/eMag)FMN~470 nm (on)Dark (off)Both components are light sensitive so as to increase the spatial precision and reduce leakiness.paCas9 for transcriptional control [89.Konermann S. et al.Optical control of mammalian endogenous transcription and epigenetic states.Nature. 2013; 500: 472-476Crossref PubMed Scopus (607) Google Scholar];PA-Cre, PA-Cre 3.0, and PA-Flp for site-specific recombination [105.Kim C.K. et al.Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions.Elife. 2019; 8e43826Crossref Scopus (27) Google Scholar, 106.Kawano F. et al.A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.Nat. Chem. Biol. 2016; 12: 1059-1064Crossref PubMed Scopus (106) Google Scholar, 107.Morikawa K. et al.Photoactivatable Cre recombinase 3.0 for in vivo mouse applications.Nat. Commun. 2020; 11: 2141Crossref PubMed Scopus (0) Google Scholar].CRY2-CIBFAD~470 nm (on)Dark (off)CRY2 mutants L348F or W349R with prolonged or shortened photocycles (t1/2, off ~24 and ~2.5 min, respectively);longer CRY2 variant (residues 1–535) and truncated CIB1 (CIBN) exhibit reduced basal association in the dark.LITE for optical control of TALEN-mediated (epi)genomic control [88.Engreitz J. et al.CRISPR Tools for systematic studies of RNA regulation.Cold Spring Harb. Perspect. Biol. 2019; 11a035386Crossref PubMed Scopus (14) Google Scholar];LACE, LACK, and CPTS for transcriptional regulation [90.Nihongaki Y. et al.Photoactivatable CRISPR-Cas9 for optogenetic genome editing.Nat. Biotechnol. 2015; 33: 755-760Crossref PubMed Scopus (400) Google Scholar, 91.Nihongaki Y. et al.CRISPR-Cas9-based photoactivatable transcription system.Chem. Biol. 2015; 22: 169-174Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 92.Nihongaki Y. et al.CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation.Nat. Methods. 2017; 14: 963-966Crossref PubMed Scopus (108) Google Scholar];PA-Cre 2.0 for optical control of Cre-mediated DNA recombination [40.Taslimi A. et al.Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase.Nat. Chem. Biol. 2016; 12: 425-430Crossref PubMed Google Scholar];PAMEC for optogenetic control of RNA modifications [113.Liu R. et al.Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins.Nat. Biotechnol. 2022; 40: 779-786Crossref PubMed Scopus (1) Google Scholar];LINTAD for inducible transgene expression [117.O'Donoghue G.P. et al.T cells selectively filter oscillatory signals on the minutes timescale.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e201985118Google Scholar].PhyA-FHY1/FHL or REDMAPPCB640–660 nm (on)740–780 (off)Red light-induced heterodimerization pairs;dissociates under far-red light;requires external addition of PCB or its analogsREDMAP for red light-inducible transcriptional programming in mammals [61.Sorokina O. et al.A switchable light-input, light-output system modelled and constructed in yeast.J. Biol. Eng. 2009; 3: 15Crossref PubMed Scopus (0) Google Scholar].PhyB-PIF3/6PCB640–660 nm (on)740–780 nm (off)Red light-induced heterodimerization;dissociates under far-red light;requires external addition of PCB or its analogsPyB-PIF3 for reconstitution of GAL4 transcription factor [121.Bracha D. et al.Mapping local and global liquid phase behavior in living cells using photo-oligomerizable seeds.Cell. 2018; 175: 1467-1480Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 122.Shimizu-Sato S. et al.A light-switchable gene promoter system.Nat. Biotechnol. 2002; 20: 1041-1044Crossref PubMed Scopus (482) Google Scholar].nanoReD (DrBphP + LDB)Biliverdin660 nm (on)780 nm (off)Nanobody-based, red light-induced heterodimerization;dissociates under far-red light.COMBINES-LID system for controlling gene expression [65.Leopold A.V. et al.Neurotrophin receptor tyrosine kinases regulated with near-infrared light.Nat. Commun. 2019; 10: 1129Crossref PubMed Scopus (41) Google Scholar].BphP+PpsR2; BphP+Q-PAS1Biliverdin780 nm (on)640 nm/dark (off)Far-red or NIR light-responsive dimerization pairs;dissociates under red light stimulation.Compatible for transcriptional regulation in vivo [66.Huang Z. et al.Creating red light-switchable protein dimerization systems as genetically encoded actuators with high specificity.ACS Synth. Biol. 2020; 9: 3322-3333Crossref PubMed Scopus (9) Google Scholar,67.Redchuk T.A. et al.Near-infrared optogenetic pair for protein regulation and spectral multiplexing.Nat. Chem. Biol. 2017; 13: 633-639Crossref PubMed Scopus (99) Google Scholar].IV. DissociationUVR8Tryptophan antennas280–315 nmHomodimerizes in the dark; reversibly monomerizes under UV light stimulation with subsequent heterodimerization with COP1UVR8-COP1 compatible for transcriptional regulation [70.Yin R. et al.Two distinct domains of the UVR8 photoreceptor interact with COP1 to initiate UV-B signaling in Arabidopsis.Plant Cell. 2015; 27: 202-213Crossref PubMed Scopus (89) Google Scholar].LOVTRAP(LOV2 + Zdk)FMN450–470 nmBlue light-induced dissociation; tunable photocycles (t1/2 from 1.7 to 496 s) with LOV2 mutants.Reversibly sequesters effector proteins, such as transcription factors, at different organelles [71.Crefcoeur R.P. et al.Ultraviolet-B-mediated induction of protein-protein interactions in mammalian cells.Nat. Commun. 2013; 4: 1779Crossref PubMed Scopus (115) Google Scholar].V. Self-cleavagePhoCl or PhoCl2fSYG triad400 nm (on)Light-inducible irreversible self-cleavage into two fragments.Design of light-cleavable Cre recombinases and transcription factors [73.Zayner J.P. Sosnick T.R. Factors that control the chemistry of the LOV domain photocycle.PLoS One. 2014; 9e87074Crossref PubMed Scopus (68) Google Scholar].VI. Photoactivable enzymesBphSPCB660 nm (on)760 nm (off)Light-triggered c-di-GMP production to activate the c-di-GMP responsive transcriptional machineryFar-red light-controlled transgene expression, genome/epigenome editing and endogenous gene activation or transcriptional programming [77.Ryu M.H. Gomelsky M. Near-infrared light responsive synthetic c-di-GMP module for optogenetic applications.ACS Synth. Biol. 2014; 3: 802-810Crossref PubMed Scopus (90) Google Scholar,78.Yu Y. et al.Engineering a far-red light-activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors.Sci. Adv. 2020; 6eabb1777Crossref Scopus (29) Google Scholar,94.He L. et al.Design of smart antibody mimetics with photosensitive switches.Adv. Biol. (Weinh.). 2021; 5e2000541Google Scholar, 95.Shao J. et al.Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E6722-E6730Crossref PubMed Scopus (0) Google Scholar, 96.Shao J. et al.Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice.Sci. Transl. Med. 2017; 9eaal2298Crossref Scopus (111) Google Scholar]. Open table in a new tab Given that opsin-based optogenetics has been extensively reviewed elsewhere [12.Rajasethupathy P. et al.Targeting neural circuits.Cell. 2016; 165: 524-534Abstract Full Text Full Text PDF PubMed Google Scholar,13.Kim C.K. et al.Integration of optogenetics with complementary methodologies in systems neuroscience.Nat. Rev. Neurosci. 2017; 18: 222-235Crossref PubMed Scopus (384) Google Scholar], we will highlight herein PSMs derived from non-opsin photoreceptors [1.Tan P. et al.Optophysiology: illuminating cell physiology with optogenetics.Physiol. Rev. 2022; 102: 1263-1325Crossref PubMed Scopus (0) Google Scholar,3.Lu X. et al.Engineering photosensory modules of non-opsin-based optogenetic actuators.Int. J. Mol. Sci. 2020; 21: 6522Crossref Scopus (8) Google Scholar], discuss generally applicable optogenetic engineering strategies, and highlight latest advances in the applications of opsin-free optogenetics for precise transcriptional programming and versatile genetic engineering. On the basis of the photochemical properties of their light-sensing moieties, PSMs commonly used for transcriptional and genetic engineering can be classified into the following major categories (Table 1) [1.Tan P. et al.Optophysiology: illuminating cell physiology with optogenetics.Physiol. Rev. 2022; 102: 1263-1325Crossref PubMed Scopus (0) Google Scholar,2.Dagliyan O. Hahn K.M. Controlling protein conformation with light.Curr. Opin. Struct. Biol. 2019; 57: 17-22Crossref PubMed Scopus (16) Google Scholar,4.Seong J. Lin M.Z. Optobiochemistry: genetically encoded control of protein activity by light.Annu. Rev. Biochem. 2021; 90: 475-501Crossref PubMed Scopus (9) Google Scholar, 5.Losi A. et al.Blue-light receptors for optogenetics.Chem. Rev. 2018; 118: 10659-10709Crossref PubMed Scopus (115) Google Scholar, 6.Zhang K. Cui B. Optogenetic control of intracellular signaling pathways.Trends Biotechnol. 2015; 33: 92-100Abstract Full Text Full Text PDF PubMed Google Scholar, 7.Toettcher J.E. et al.The promise of optogenetics in cell biology: interrogating molecular circuits in space and time.Nat. Methods. 2011; 8: 35-38Crossref PubMed Scopus (181) Google Scholar, 8.Tan P. et al.Optogenetic immunomodulation: shedding light on antitumor immunity.Trends Biotechnol. 2017; 35: 215-226Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]: (i) plant UVR8 with tryptophan residues as the putative photo-sensing antenna (280–315 nm); (ii) plant cryptochrome (CRY) or light-oxygen-voltage (LOV) domain with flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) (450–470 nm) as the light-absorbing cofactors; (iii) bacterial photoactive yellow protein (PYP) with p-coumaric acid (or 4-hydroxycinnamic acid) as the cofactor (450 nm); (iv) microbial or vertebrate opsins bearing retinal or vitamin A derivatives (450–700 nm); (v) DronpaN with a Cys-Tyr-Gly (CYG) triad as the chromophore (400–500 nm); (vi) plant and bacterial phytochromes that utilize phycocyanobilin (PCB), biliverdin (BV), or their analogs (640–780 nm) as the photon-sensing antennas. Given that light emitting in the longer wavelength tends to penetrate deeper in living tissues, red-shifted PSMs in the far-red and near-infrared radiation (NIR) range are more compatible with in vivo applications by obviating the need of optical fiber or micro-LED implantation. To overcome the limited depth of tissue penetration associated with most existing blue/green light-activatable photoreceptors, researchers have developed upconversion nanoparticles (UCNPs) as nano-transducers to convert deep tissue-penetrating NIR light into blue light for photostimulation [14.He L. et al.Near-infrared photoactivatable control of Ca(2+) signaling and optogenetic immunomodulation.Elife. 2015; 4e10024Crossref Google Scholar, 15.He L. et al.Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering.Nat. Chem. Biol. 2021; 17: 915-923Crossref PubMed Scopus (7) Google Scholar, 16.He L. et al.Optogenetic control of non-apoptotic cell death.Adv. Sci. (Weinh.). 2021; 82100424Google Scholar, 17.Yu N. et al.Near-infrared-light activatable nanoparticles for deep-tissue-penetrating wireless optogenetics.Adv. Healthc. Mater. 2019; 8e1801132Crossref Scopus (51) Google Scholar, 18.Nguyen N.T. et al.Nano-optogenetic engineering of CAR T cells for precision immunotherapy with enhanced safety.Nat. Nanotechnol. 2021; 16: 1424-1434Crossref PubMed Scopus (5) Google Scholar, 19.Chen S. et al.Near-infrared deep brain stimulation via upconversion nanoparticle-mediated optogenetics.Science. 2018; 359: 679-684Crossref PubMed Scopus (609) Google Scholar]. With this upconversion nano-optogenetic approach, the depth of tissue penetration can be remarkably improved from 0.5~1 mm at 470 nm to up to centimeters at 980 nm. Furthermore, luciferases-catalyzed bioluminescence can be exploited to enable optogenetic stimulation in deeply buried tissues [16.He L. et al.Optogenetic control of non-apoptotic cell death.Adv. Sci. (Weinh.). 2021; 82100424Google Scholar,20.Berglund K. et al.Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E358-E367Crossref PubMed Google Scholar], albeit at the cost of partially sacrificing the spatial resolution. Transcriptional programming and genome editing are among the most pursued research directions in the synthetic biology and genetics fields. The intricate processes from gene transcription to mRNA translation provide ample opportunities for optogenetic intervention. Not surprisingly, a myriad of optogenetic devices have been crafted to control the cellular flow of information from DNA to RNA to protein, as described in the central dogma. Here, we briefly highlight modular strategies that are commonly applied to exert optical control over gene regulation (Figure 1). Genetically encoded optogenetic modules, such as bacterial PYP and plant LOV, are often used as fusion tags to mask the active sites within a protein of interest via steric hindrance. Alternatively, these compact photoswitches (<150 residues) can be modularly inserted into sensitive spots that are distant from the active sites of host proteins, thereby exerting allosteric regulation in a light-dependent manner (Figure 1 and Box 1) [21.Dagliyan O. et al.Engineering extrinsic disorder to control protein activity in living cells.Science. 2016; 354: 1441-1444Crossref PubMed Scopus (136) Google Scholar].Box 1Genetically encoded allosteric photoswitchesLOVLOV belongs to the Per-ARNT-Sim (PAS) superfamily of proteins that sense environmental stimuli, such as oxygen, redox state, light, and voltage, and are capable of triggering changes in protein structures to affect downstream sensor–effector interactions [5.Losi A. et al.Blue-light receptors for optogenetics.Chem. Rev. 2018; 118: 10659-10709Crossref PubMed Scopus (115) Google Scholar,22.Glantz S.T. et al.Functional and topological diversity of LOV domain photoreceptors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E1442-E1451Crossref PubMed Scopus (90) Google Scholar,23.Hart J.E. Gardner K.H. Lighting the way: recent insights into the structure and regulation of phototropin blue light receptors.J. Biol. Chem. 2021; 296100594Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar]. A typical LOV domain contains a central PAS core and two alpha-helices (A′a and Jα). In Avena sativa LOV2 (AsLOV2), an effector domain fused directly after the C terminus of Jα helix can be caged by the PAS core in the dark, whereas blue light stimulation (450–470 nm) initiates the formation of a photo-adduct between the cofactor FMN (abundantly available in mammalian cells) and a nearby cysteine, thereby causing conformational changes to unfold the distal Jα helix with subsequent effector uncaging (Figure 1A) [24.Yao X. et al.Estimation of the available free energy in a LOV2-J alpha photoswitch.Nat. Chem. Biol. 2008; 4: 491-497Crossref PubMed Scopus (110) Google Scholar,25.Wu Y.I. et al.A genetically encoded photoactivatable Rac controls the motility of living cells.Nature. 2009; 461: 104-108Crossref PubMed Scopus (811) Google Scholar]. Interestingly, circularly permuted LOV2 (cpLOV2) still retains its photo-responsiveness and can also be used as a modular photoswitch for optogenetic engineering [15.He L. et al.Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering.Nat. Chem. Biol. 2021; 17: 915-923Crossref PubMed Scopus (7) Google Scholar]. The photocycles of LOV domains sustain from milliseconds to minutes or hours, and mutations can be further introduced to diversity the kinetic properties [26.Pudasaini A. et al.LOV-based optogenetic devices: light-driven modules to impart photoregulated control of cellular signaling.Front. Mol. Biosci. 2015; 2: 18Crossref PubMed Google Scholar]. AsLOV2-based tools are widely used in the control of gene expression and genome engineering (Table 1).PYPThe engineering of PYP-based photoswitch relies on the folded state of PYP to sterically block the interaction between fused protein moiety and its targeting molecules or binding partners (Figure 1A). In the darkness, the chromophore p-coumaric acid adopts a trans configuration and PYP remains well folded. Upon blue light stimulation, the chromophore isomerizes to the cis configuration and PYP undergoes conformational changes, with its N-terminal region (residues 1–25) almost completely unfolded [27.Ramachandran P.L. et al.The short-lived signaling state of the photoactive yellow protein photoreceptor revealed by combined structural probes.J. Am. Chem. Soc. 2011; 133: 9395-9404Crossref PubMed Scopus (65) Google Scholar,28.Pande K. et al.Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein.Science. 2016; 352: 725-729Crossref PubMed Scopus (287) Google Scholar]. Taking advantage of the large light-inducible conformational changes, PYP was fused with GCN4-bZIP to cage the leucine zipper dimerization domain and block DNA binding in the darkness. Light stimulation overcomes the caging effect to restore GCN4-bZIP dimerization and subsequent target DNA binding [29.Morgan S.A. et al.Structure-based design of a photocontrolled DNA binding protein.J. Mol. Biol. 2010; 399: 94-112Crossref PubMed Scopus (42) Google Scholar,30.Morgan S.A. Woolley G.A. A photoswitchable DNA-binding protein based on a truncated GCN4-photoactive yellow protein chimera.Photochem. Photobiol. Sci. 2010; 9: 1320-1326Crossref PubMed Scopus (29) Google Scholar]. Adopting a similar design principle, a light-dependent dominant negative inhibitor of the TF CREB (opto-DN-CREB) was created. Blue light stimulation drives the conformational change in PYP, which in turn sequesters an engineered acidic-CREB (A-CREB) and prevents coiled-coil formation between A-CREB and CREB, thereby permitting normal CREB dimerization and DNA binding [31.Ali A.M. et al.Optogenetic inhibitor of the transcription factor CREB.Chem. Biol. 2015; 22: 1531-1539Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. One caveat associated with PYP is the requirement of an exogenous cofactor p-coumaric acid, which is absent in mammalian cells and thus may hamper its applications in mammals. LOV LOV belongs to the Per-ARNT-Sim (PAS) superfamily of proteins that sense environmental stimuli, such as oxygen, redox state, light, and voltage, and are capable of triggering changes in protein structures to affect downstream sensor–effector interactions [5.Losi A. et al.Blue-light receptors for optogenetics.Chem. Rev. 2018; 118: 10659-10709Crossref PubMed Scopus (115) Google Scholar,22.Glantz S.T. et al.Functional and topological diversity of LOV domain photoreceptors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E1442-E1451Crossref PubMed Scopus (90)