Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase

Taslimi, Amir; Zoltowski, Brian D.; Miranda, Jose G.; Pathak, Gopal P.; Hughes, Robert M.; Tucker, Chandra L.

doi:10.1038/nchembio.2063

Cited by 218 publications

(285 citation statements)

References 52 publications

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“…Later, its natural oligomerization ability was used in optogenetic clustering approaches 9 . Further tuning of the engineered light-activatable systems led to a design of the new generation of dimerizers for advanced control of the protein localization 10 , cell signaling 11 and recombinase activity 12 . All these optogenetic systems sense 440–480 nm light.…”

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confidence: 99%

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

et al. 2017

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Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1–PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix–PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1–Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1–Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.

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confidence: 99%

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

et al. 2017

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“…However, this system has potential drawbacks. First, the dissociation reaction has slow kinetics and is uncontrollable, though faster dissociation mutants of the CRY2-CIB1 pair have been reported recently (15). Second, the use of blue light for photoactivation of the CRY2-CIB1 is not compatible with fluorescence imaging with GFP-based biosensors such as FRET biosensors (16,17).…”

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confidence: 99%

Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling

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Oda

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

114

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Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)–phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB–PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB–PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB–PIF system, which paves the way for its application to a living animal.

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“…However, systems that might be sensitive to low transgene expression levels in the “off” state, such as angiogenic sprouting, should use CIB1 N -Cre C . Moreover, next-generation versions of the light-inducible Cre recombinase may facilitate even greater control over levels of induced recombination and downstream effects 58 . Therefore the options for tight control and flexible dynamic range of the system, coupled with the ability to induce sustained effects with transient stimuli in a spatiotemporally controlled fashion, will enable many applications in tissue engineering, synthetic biology, gene therapy, and basic science.…”

Section: Discussionmentioning

confidence: 99%

An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis

et al. 2017

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The precise spatial and temporal control of gene expression, cell differentiation, and tissue morphogenesis has widespread application in regenerative medicine and the study of tissue development. In this work, we applied optogenetics to control cell differentiation and new tissue formation. Specifically, we engineered an optogenetic “on” switch that provides permanent transgene expression following a transient dose of blue light illumination. To demonstrate its utility in controlling cell differentiation and reprogramming, we incorporated an engineered form of the master myogenic factor MyoD into this system in multipotent cells. Illumination of cells with blue light activated myogenic differentiation, including upregulation of myogenic markers and fusion into multinucleated myotubes. Cell differentiation was spatially patterned by illumination of cell cultures through a photomask. To demonstrate the application of the system to controlling in vivo tissue development, the light inducible switch was used to control the expression of VEGF and angiopoietin-1, which induced angiogenic sprouting in a mouse dorsal window chamber model. Live intravital microscopy showed illumination-dependent increases in blood-perfused microvasculature. This optogenetic switch is broadly useful for applications in which sustained and patterned gene expression is desired following transient induction, including tissue engineering, gene therapy, synthetic biology, and fundamental studies of morphogenesis.

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Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase

Cited by 218 publications

References 52 publications

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

Near-infrared optogenetic pair for protein regulation and spectral multiplexing

Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling

An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis

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