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Homing endonucleases are used in a wide range of biotechnological applications including gene editing, in gene drive systems, and for the modification of DNA structures, arrays, and prodrugs. However, controlling nuclease activity and sequence specificity remain key challenges when developing new tools. Here a photoresponsive homing endonuclease was engineered for optical control of DNA cleavage by partitioning DNA binding and nuclease domains of the monomeric homing endonuclease I-TevI into independent polypeptide chains. Use of the Aureochrome1a light-oxygen-voltage domain delivered control of dimerization with light. Illumination reduced the concentration needed to achieve 50% cleavage of the homing target site by 6-fold when compared to the dark state, resulting in an up to 9-fold difference in final yields between cleavage products. I-TevI nucleases with and without a native I-TevI zinc finger motif displayed different nuclease activity and sequence preference impacting the promiscuity of the nuclease domain. By harnessing an alternative DNA binding domain, target preference was reprogrammed only when the nuclease lacked the I-TevI zinc finger motif. This work establishes a first-generation photoresponsive platform for spatiotemporal activation of DNA cleavage.

We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ~ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (~1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit. [ABSTRACT FROM AUTHOR]

Optogenetics harnesses natural photoreceptors to non-invasively control selected processes in cells with previously unmet spatiotemporal precision. Linking the activity of a protein of choice to the conformational state of a photosensor domain through allosteric coupling represents a powerful method for engineering light-responsive proteins. It enables the design of compact and highly potent single-component optogenetic systems with fast on- and off-switching kinetics. However, designing protein-photoreceptor chimeras, in which structural changes of the photoreceptor are effectively propagated to the fused effector protein, is a challenging engineering problem and often relies on trial and error. Here, recent advances in the design and application of optogenetic allosteric switches are reviewed. First, an overview of existing optogenetic tools based on inducible allostery is provided and their utility for cell biology applications is highlighted. Focusing on light-oxygen-voltage domains, a widely applied class of small blue light sensors, the available strategies for engineering light-dependent allostery are presented and their individual advantages and limitations are highlighted. Finally, high-throughput screening technologies based on comprehensive insertion libraries, which could accelerate the creation of stimulus-responsive receptor-protein chimeras for use in optogenetics and beyond, are discussed., (© 2020 The Authors. Advanced Biology published by Wiley-VCH GmbH.)

Protein trans-splicing is a posttranslational modification that joins two protein fragments together via a peptide a bond in a process that does not require exogenous cofactors. Towards achieving cellular control, synthetically engineered systems have used a variety of stimuli such as small molecules and light. Recently, split inteins have been engineered to be photoactive by the LOV2 domain (named LOVInC). Herein, we discuss (1) designing of LOV2-activated target proteins (e.g., inteins), (2) selecting feasible splice sites for the extein, and (3) imaging cells that express LOVInC-based target exteins.

We have developed an optogenetic technique for the activation of diaphanous-related formins. Our approach is based on fusion of the light-oxygen-voltage 2 domain of Avena sativa Phototrophin1 to an isolated Diaphanous Autoregulatory Domain from mDia1. This "caged" diaphanous auto-regulatory domain was inactive in the dark but in the presence of blue light rapidly activated endogenous diaphanous-related formins. Using an F-actin reporter, we observed filopodia and lamellipodia formation as well as a steady increase in F-actin along existing stress fibers, starting within minutes of photo-activation. Interestingly, we did not observe the formation of new stress fibers. Remarkably, a 1.9-fold increase in F-actin was not paralleled by an increase in myosin II along stress fibers and the amount of tension generated by the fibers, as judged by focal adhesion size, appeared unchanged. Our results suggest a decoupling between F-actin accumulation and contractility in stress fibers and demonstrate the utility of photoactivatable diaphanous autoregulatory domain for the study of diaphanous-related formin function in cells., (Copyright © 2013 Wiley Periodicals, Inc.)