Building a Simple and Versatile Illumination System for Optogenetic Experiments.

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

Engineering light-controllable CAR T cells for cancer immunotherapy.

T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.

An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics.

While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre(2-59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC(60-343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.

Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells.