Curated Optogenetic Publication Database

Abstract: 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.

Abstract: Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. In previous work, we used cryptochrome 2 (CRY2) and CIB1, Arabidopsis proteins that interact upon light illumination, to regulate transcription with light in yeast. While adopting this approach to regulate transcription in mammalian cells, we observed light-dependent redistribution and clearing of CRY2-tethered proteins within the nucleus. The nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within the CRY2-fused transcriptional activators. We used this knowledge to develop two different approaches to regulate cellular protein levels with light: a system using CRY2 and CIB1 to induce protein expression with light through stimulation of transcription, and a system using CRY2 and a LOV-fused degron to simultaneously block transcription and deplete protein levels with light. These tools will allow precise, bi-directional control of gene expression in a variety of cells and model systems.

Abstract: Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR-Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.

Abstract: The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells.

Abstract: Advanced gene regulatory systems are necessary for scientific research, synthetic biology, and gene-based medicine. An ideal system would allow facile spatiotemporal manipulation of gene expression within a cell population that is tunable, reversible, repeatable, and can be targeted to diverse DNA sequences. To meet these criteria, a gene regulation system was engineered that combines light-sensitive proteins and programmable zinc finger transcription factors. This system, light-inducible transcription using engineered zinc finger proteins (LITEZ), uses two light-inducible dimerizing proteins from Arabidopsis thaliana, GIGANTEA and the LOV domain of FKF1, to control synthetic zinc finger transcription factor activity in human cells. Activation of gene expression in human cells engineered with LITEZ was reversible and repeatable by modulating the duration of illumination. The level of gene expression could also be controlled by modulating light intensity. Finally, gene expression could be activated in a spatially defined pattern by illuminating the human cell culture through a photomask of arbitrary geometry. LITEZ enables new approaches for precisely regulating gene expression in biotechnology and medicine, as well as studying gene function, cell-cell interactions, and tissue morphogenesis.