Biological processes are naturally regulated with high spatial and temporal control, as is perhaps most evident in metazoan embryogenesis Chemical tools have been extensively utilized in cell and developmental biology to investigate cellular processes, and conditional control methods have expanded applications of these technologies toward resolving complex biological questions Light represents an excellent external trigger since it can be controlled with very high spatial and temporal precision To this end, several optically regulated tools have been developed and applied to living systems In this review we discuss recent developments of optochemical tools, including small molecules, peptides, proteins, and nucleic acids that can be irreversibly or reversibly controlled through light irradiation, with a focus on applications in cells and animals

Optochemical Control of Biological Processes in Cells and Animals.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either boceprevir or telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro, with 50% inhibitory concentration values ranging from 7.6 to 748.5 nM. The cocrystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a transgenic mouse model of SARS-CoV-2 infection, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.

https://science.sciencemag.org/content/sci/early/2021/02/17/science.abf1611.full.pdf

SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse model

Genome editing technologies, such as CRISPR/Cas9, are promising for treating otherwise incurable genetic diseases. Great progress has been made for ex vivo genome editing; however, major bottlenecks exist in the development of efficient, safe, and targetable in vivo delivery systems, which are needed for the treatment of many diseases. To achieve high efficacy and safety in therapeutic in vivo genome editing, editing activities must be controlled spatially and temporally in the body, which requires novel materials, delivery strategies, and control mechanisms. Thus, there is currently a tremendous opportunity for the biomaterials research community to develop in vivo delivery systems that overcome the problems of low editing efficiency, off-targeting effect, safety, and cell and tissue specificity. In this Review, we summarize delivery approaches and provide perspectives on the challenges and possible solutions, aiming to stimulate further development of engineered materials for in vivo delivery of genome-editing machinery.

Engineered materials for in vivo delivery of genome-editing machinery.

Photoactivated materials have found widespread use in biological and medical applications and are playing an increasingly important role in the nascent field of three-dimensional (3D) bioprinting. Light can be used as a trigger to drive the formation or the degradation of chemical bonds, leading to unprecedented spatiotemporal control over a material's chemical, physical, and biological properties. With resolution and construct size ranging from nanometers to centimeters, light-mediated biofabrication allows multicellular and multimaterial approaches. It promises to be a powerful tool to mimic the complex multiscale organization of living tissues including skin, bone, cartilage, muscle, vessels, heart, and liver, among others, with increasing organotypic functionality. With this review, we comprehensively discuss photochemical reactions, photoactivated materials, and their use in state-of-the-art deposition-based (extrusion and droplet) and vat polymerization-based (one- and two-photon) bioprinting. By offering an up-to-date view on these techniques, we identify emerging trends, focusing on both the chemistry and instrument aspects, thereby allowing the readers to select the best-suited approach. Starting with photochemical reactions and photoactivated materials, we then discuss principles, applications, and limitations of each technique. With a critical eye to the most recent achievements, the reader is guided through this exciting, emerging field, with special emphasis on cell-laden hydrogel constructs.

Guiding Lights: Tissue Bioprinting Using Photoactivated Materials.

Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

A light- and calcium-gated transcription factor for imaging and manipulating activated neurons

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitc...

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription

An important goal in synthetic biology is to engineer biochemical pathways to address unsolved biomedical problems. One long-standing problem in molecular medicine is the specific identification and ablation of cancer cells. Here, we describe a method, named Rewiring of Aberrant Signaling to Effector Release (RASER), in which oncogenic ErbB receptor activity, instead of being targeted for inhibition as in existing treatments, is co-opted to trigger therapeutic programs. RASER integrates ErbB activity to specifically link oncogenic states to the execution of desired outputs. A complete mathematical model of RASER and modularity in design enable rational optimization and output programming. Using RASER, we induced apoptosis and CRISPR-Cas9-mediated transcription of endogenous genes specifically in ErbB-hyperactive cancer cells. Delivery of apoptotic RASER by adeno-associated virus selectively ablated ErbB-hyperactive cancer cells while sparing ErbB-normal cells. RASER thus provides a new strategy for oncogene-specific cancer detection and treatment.

A compact synthetic pathway rewires cancer signaling to therapeutic effector release.

The coronavirus main protease, Mpro, is a key protein in the virus life cycle and a major drug target. Based on crystal structures of SARSCoV2 Mpro complexed with peptidomimetic inhibitors, we recognized a binding characteristic shared with proline-containing inhibitors of hepatitis C virus protease. Initial tests showed that this subclass of HCV protease inhibitors indeed exhibited activity against Mpro. Postulating a benefit for a preorganized backbone conformation, we designed new ketoamide-based Mpro inhibitors based on central proline rings. One of the designed compounds, ML1000, inhibits Mpro with low-nanomolar affinity and suppresses SARSCoV2 viral replication in human cells at sub-micromolar concentrations. Our findings identify ML1000 as a promising new pre-organized scaffold for the development of anti-coronavirus drugs.

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Xinzhi Zou

Papers

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription

A compact synthetic pathway rewires cancer signaling to therapeutic effector release.

Rational design of a new class of protease inhibitors for the potential treatment of coronavirus diseases