Fluorescent proteins and small-molecule dyes offer complementary advantages for biological imaging: Proteins are amenable to genetic tagging, whereas dyes provide superior brightness and photostability. To combine these strengths, we used de novo protein design to generate small, nanomolar-affinity, high-selectivity binders (NovoTags) for three cell-permeable dyes spanning the visible spectrum. We show that the NovoTag fluorescent lifetimes can be tuned and demonstrate their application in lifetime- and wavelength-based multiplexed fluorescence imaging. We also designed a two-chain version (NovoSplit) that functions as a chemically induced dimerization system with fluorescent readout in living cells or as a minimally perturbing proximity probe in fixed cells. Our approach combines the advantages of fluorescent proteins and small-molecule dyes, thus expanding the toolkit for cellular imaging.

Molecular recording is an emerging paradigm for measuring biology over time. Enhancer-mediated genomic recording of activity in multiplex (ENGRAM) is a recently described synthetic biology circuit architecture that converts the transient activity of cis-regulatory elements (CREs) into stable genomic records that can be retrospectively recovered via DNA sequencing. Here we provide a step-by-step protocol for conducting ENGRAM experiments and analyzing the resulting data. We also describe key design considerations for ENGRAM recorders, summarize the strengths and limitations of ENGRAM, and highlight applications, including multiplex signal recording and high-throughput CRE screening. In contrast to other systems for DNA-based recording in mammalian systems, ENGRAM relies on prime editing-mediated insertions to record the activity of a given CRE, such that it is inherently multiplexable-for example, four-base-pair insertions can represent the activities of up to 256 distinct CREs. A further contrast lies with ENGRAM's compatibility with DNA Typewriter, which facilitates the capture of signal order. For users with basic skills in molecular biology, mammalian cell culture and DNA sequencing analysis, ENGRAM experiments can typically be completed within 5-6 weeks.

Protein kinases are critical regulators of cellular signaling, but precise modulation of their activity remains challenging due to their high structural conservation. Here, we present de novo designed genetically encoded miniproteins capable of activating or inhibiting focal adhesion kinase (FAK) by directly targeting the kinase domain itself. Among 96 binders designed to stabilize distinct conformational states of FAK, 33 modulated kinase activity. Biochemical characterization of the four most potent modulators revealed that two designs inhibit FAK with low-nanomolar IC 50 values while the remaining two potentiated FAK activity by more than two-fold. When expressed in cells, the modulators preserved the same inhibitory and activating effects observed in vitro, establishing that designed conformational binders can directly tune FAK signaling in living cells. Taking advantage of the high similarity between kinases, we redesigned the FAK inhibitors to inhibit Src kinase. Our approach establishes a versatile platform for selective and genetically encoded kinase control as a way to rewire cell signaling and as a starting point for the discovery of novel modulatory sites of kinases.

Quasisymmetric icosahedral viral capsids achieve larger sizes than possible with strictly symmetric icosahedra by tessellating pentagons and hexagons using a single subunit that adopts different conformations in symmetrically non-equivalent locations1,2. Recapitulating such quasisymmetric architectures through computational design is a considerable challenge in nanomaterials engineering. Here we introduce a computational design strategy based on geometric frustration to generate two-component, quasisymmetric protein cages with customizable properties. We designed complementary trimeric and dimeric protein components that co-assemble into positively curved local hexagonal assemblies. Hexagonal lattices cannot tile spherical surfaces; instead, the components form closed sphere-like cage assemblies through incorporation of curvature-inducing pentagonal defects, as evidenced by electron microscopy. By designing dimers that encode different local curvatures, we programmed cage dimensions ranging from 40 to over 200 nm in diameter and with molecular weights from 2 MDa to over 50 MDa, comparable with natural virus capsids. We further functionalized these large cages with additional protein domains to enable ribonucleoprotein cargo loading and cellular uptake. Fluorescently labelled cage assemblies expressed in mammalian cells function as rheological probes and cargo recruiters, enabling a systematic study of size-dependent cytoplasmic diffusion and protein localization. Thus, the quasi-symmetry that has long fascinated structural biologists can now be achieved by computational protein design, with immediate applications to biologics delivery and molecular cell biology.

Neural activity in the delta range (1.0-4.5 Hz) during non-rapid eye movement (NREM) sleep is crucial for brain plasticity and overall brain health. Recent research has shown that changes in NREM delta activity can occur locally, and activity can vary across different brain regions. Ischemic stroke results in focal brain injury and long-term disability. While sleep disruption during the acute phase of stroke is known to hinder recovery, the relationship between region-specific changes in NREM delta activity and functional recovery remains poorly understood. To investigate these localized changes in NREM delta activity with high spatial resolution, we utilized wide-field optical imaging (WFOI) in mice that expressed GCaMP6f, a fluorescent calcium indicator, in cortical excitatory pyramidal neurons. Sleep was longitudinally recorded before and at 24 hours, one week, and four weeks after photothrombotic stroke in the left somatosensory forepaw cortex. In the acute phase of stroke (24 hours post stroke), mice exhibited decreased delta activity in the infarct and peri-infarct regions during NREM sleep. Increased delta activity in the contralesional hemisphere and decreased delta activity in the perilesional region during NREM sleep in the acute phase were associated with poor behavioral recovery, as measured by performance on the cylinder rearing test. These findings suggest that region-specific NREM delta activity may play a crucial role in stroke recovery and warrant further investigation to determine whether modulating delta activity in targeted brain areas during NREM sleep could aid recovery.

Mammalian stem cells possess a remarkable capacity for self-organization, a property that underlies increasingly sophisticated in vitro models of early development. However, even under carefully controlled conditions, stem cell-derived models exhibit substantial "inter-individual" heterogeneity. Focusing on gastruloids, a powerful model of the early posterior embryo1, we sought to investigate the origins of this heterogeneity. To this end, we developed a scalable protocol for generating gastruloids that are monoclonal, i.e. derived from a single mouse embryonic stem cell (mESC). Single cell transcriptional profiling of monoclonal gastruloids revealed extensive inter-individual heterogeneity, with some hardly progressing, others resembling conventional gastruloids but biased towards mesodermal or neural lineages, and yet others bearing cell types rare or absent from conventional polyclonal gastruloids. To investigate this further, we leveraged DNA Typewriter2 to record the cell lineage relationships among the mESCs from which monoclonal gastruloids originate. Early in the expansion of "founder" mESCsーprior to induction of the resulting aggregates to form gastruloidsーwe observe clear examples of fate bias or fate restriction, i.e. sister clades that exhibit markedly different cell type compositions. In a separate experiment with DNA Typewriter, we reconstructed a monophyletic "tree of trees", composed of ~50,000 cells derived from ~100 gastruloids, all descended from a single "founder of founders" stem cell. From these data, we find that founder mESCs that are more closely related are more likely to give rise to monoclonal gastruloids with similar cell type compositions. Our results suggest that fluctuations in the intrinsic states of mESCs are heritable, and shape their descendants' fates across many cell divisions. Our study also showcases how DNA Typewriter can be used to reconstruct high-resolution, monophyletic cell lineage trees in stem cell models of early development.

Advances in precise genome editing are enabling genomic recordings of cellular events. Since the initial demonstration of CRISPR-based genome editing, the field of genomic recording has witnessed key strides in lineage recording, where clonal lineage relationships among cells are indirectly recorded as syn-thetic mutations. However, methods for directly recording and reconstructing past cellular events are still limited, and their potential for revealing new insights into cell fate decisions has yet to be realized. The field needs new sensing mod-ules and genetic circuit architectures that faithfully encode past cellular states into genomic DNA recordings to achieve such goals. Here we review recently developed strategies to construct diverse sensors and explore how emerging synthetic biology tools may help to build molecular circuits for genomic record-ing of diverse cellular events.

Sequence-specific DNA-binding proteins (DBPs) have critical roles in biology and biotechnology and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize short specific target sequences through interactions with bases in the major groove and use this method to generate binders for five distinct DNA targets with mid-nanomolar to high-nanomolar affinities. The individual binding modules have specificity closely matching the computational models at as many as six base-pair positions and higher-order specificity can be achieved by rigidly positioning the binders along the DNA double helix using RFdiffusion. The crystal structure of a designed DBP-target site complex is in close agreement with the design model and the designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method provides a route to small and, hence, readily deliverable sequence-specific DBPs for gene regulation and editing.

A standardized ‘parts list’ of sequences for genetic engineering of microbes has been indispensable to progress in synthetic biology, but few analogous parts exist for mammalian systems. Here we design libraries of extant, ancestral, mutagenized or miniaturized variants of polymerase III promoters and guide RNA (gRNA) scaffolds and quantify their abilities to mediate precise edits to the mammalian genome through multiplex prime editing. We identify thousands of parts for reproducible editing in human and mouse cell lines, including hundreds with greater activity than commonly used sequences. Saturation mutagenesis screens identify tolerated sequence variants that further enhance sequence diversity. In an application to molecular recording, we design a ‘ten key’ array that, in mammalian cells, achieves balanced activity of pegRNAs as predicted by the activity of the component parts. The data reported here will aid the design of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity for applications in multiplexed perturbation, biological recorders and complex genetic circuits.

One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here we demonstrate a strategy termed P3 editing, which links p rotein- p rotein p roximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically-induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells.

Cytokines signal by bringing receptor subunits together, but the role of receptor geometry in shaping signaling remains unclear because natural ligands enforce fixed assemblies. Here, we present a de novo protein design platform that rigidly scaffolds receptor-binding domains into defined spatial arrangements. Applying this across IL-7, type I and III interferons, IL-10, gp130, β common, and synthetic receptor pairs, we show that by varying geometry, we can bias pSTAT pathway usage and tune functional outcomes. Geometric control allowed us to decouple pSTAT1 from pSTAT5 in IL-7, separate antigen presentation (MHC-I) from checkpoint induction (PD-L1) in type I interferons, and suppress pro-inflammatory IFNγ secretion while retaining anti-inflammatory activity in IL-10. We further created minimal IL-6 and IL-3 agonists and strengthened synthetic receptor pairings inaccessible with present cytokines. These results establish receptor geometry as a central determinant of cytokine activity and provide a platform for programmable immune modulation.

BACKGROUND:Stroke is a leading cause of long-term adult disability. Behavioral testing with animal stroke models, which offers a way to evaluate the effectiveness of new interventions, currently relies on methods that are time- and labor-intensive. Automated behavioral assessments of locomotion and gait have been proposed as an alternative, but it is currently unknown whether they are sensitive enough to assess behavioral deficits following stroke of the forepaw somatosensory cortex. The purpose of this study was to compare a validated, manually assessed behavioral test, cylinder rearing (a measure of forepaw asymmetry during exploration), with automated behavior tests of locomotion in a rodent photothrombotic stroke model. METHODS:We induced a focal photothrombotic stroke in young (12-16 week old) male mice over the left forepaw somatosensory cortex, conducted behavioral testing at acute (48 h) and sub-acute (4 weeks) time points post-stroke, and then correlated behavior deficits to histological measures. RESULTS:Three automated behavioral tests were used in comparison to cylinder rearing: CatWalk (spontaneous gait), DigiGait (forced treadmill locomotion), and open field (a measure of general locomotor activity). Cylinder rearing testing showed significant forepaw asymmetry between stroke and sham groups acutely and sub-acutely after stroke. Catwalk, DigiGait, and open field tests showed no significant differences between groups. When correlating behavior to histological measures of stroke, the presence of secondary thalamic injury (STI) was associated with forepaw asymmetry on cylinder rearing. CONCLUSIONS:These findings illustrate the need to find alternative automated behavioral measures for mouse photothrombotic stroke of the forepaw somatosensory cortex.

Sleep disturbances are associated with the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and primary tauopathies. Here we demonstrate that administration of the dual orexin receptor antagonist lemborexant in the P301S/E4 mouse model of tauopathy improves tau-associated impairments in sleep-wake behavior. It also protects against chronic reactive microgliosis and brain atrophy in male P301S/E4 mice by preventing abnormal phosphorylation of tau. These neuroprotective effects in males were not observed after administration of the nonorexinergic drug zolpidem that similarly promoted nonrapid eye movement sleep. Furthermore, both genetic ablation of orexin receptor 2 and lemborexant treatment reduced wakefulness and decreased seeding and spreading of phosphorylated tau in the brain of wild-type mice. These findings raise the therapeutic potential of targeting sleep by orexin receptor antagonism to prevent abnormal tau phosphorylation and limit tau-induced damage.

Intrinsically disordered proteins and peptides play key roles in biology, but a lack of defined structures and high variability in sequence and conformational preferences have made targeting such systems challenging. We describe a general approach for designing proteins that bind intrinsically disordered protein regions in diverse extended conformations with side chains fitting into complementary binding pockets. We used the approach to design binders for 39 highly diverse unstructured targets, including polar targets, and obtained designs with 100-picomolar to 100-nanomolar affinities in 34 cases, testing ~22 designs per target. The designs function in cells and as detection reagents and are specific for their intended targets in all-by-all binding experiments. Our approach is a major step toward a general solution to the intrinsically disordered protein and peptide recognition problem.

Sleep disturbances are associated with the pathogenesis of neurodegenerative diseases including Alzheimer’s disease (AD) and primary tauopathies. We have previously shown that APOE4, the strongest genetic risk factor for AD, directly influences the severity of key pathological hallmarks of neurodegeneration including tau deposition, microglial reactivity and brain atrophy. Sleep loss influences tau accumulation and microglial reactivity in both mice and humans, suggesting that sleep loss may contribute to neurodegeneration not only by influencing protein aggregation, but also through an immune mechanism. Therefore, we aimed to investigate whether promoting sleep as a therapeutic strategy could mitigate the damaging effects of chronic microglial reactivity that contribute to tau-mediated neurodegeneration. We used lemborexant, a dual orexin receptor antagonist that promotes sleep in both mice and humans. We orally gavaged P301S/APOE4 mice, a model of tauopathy with brain atrophy, and non-tau depositing APOE4 knock-in mice daily with 30mg/kg lemborexant or vehicle (n = 16-20/genotype and treatment group) at one-hour post-dark onset. Mice were treated from 7.5 months (M), when tau-mediated neuroinflammation is observed without overt neuronal loss in P301S/APOE4 mice, until 9.5M. In P301S/APOE4 mice, lemborexant not only improved tau-associated sleep loss, specifically non-rapid eye movement sleep, but also dramatically reduced pathological tau deposition. Antagonizing orexin receptor signaling improved tau-mediated neurodegeneration noted by a decrease in plasma neurofilament light chain levels, as well as brain atrophy compared to vehicle-treated P301S/APOE4 controls. In support of these findings, lemborexant-treated P301S/APOE4 mice displayed reduced microglial reactivity of disease-associated microglia including immunostaining for CD68 and Clec7a compared to controls. Both astroglial and microglial APOE co-localization were significantly reduced in lemborexant-treated P301S/APOE4 mice, the latter of which is more commonly observed during elevated inflammatory and damaging conditions. Unbiased transcriptome profiling provided potential mechanistic insights into functional pathways influenced by lemborexant in P301S/APOE4 mice, including those regulating synaptic activity such as Slc17a7, Shank1, Shank2 , which was further accompanied by reduced pre- and post-synaptic loss. Our study provides novel therapeutic evidence that antagonizing the orexin signaling pathway using lemborexant is neuroprotective by restoring sleep deficits as well as limiting tau-mediated neuronal and synaptic damage, potentially by suppressing chronic neuroinflammation.

BACKGROUND:Wide-field calcium imaging (WFCI) with genetically encoded calcium indicators allows for spatiotemporal recordings of neuronal activity in mice. When applied to the study of sleep, WFCI data are manually scored into the sleep states of wakefulness, non-REM (NREM) and REM by use of adjunct EEG and EMG recordings. However, this process is time-consuming, invasive and often suffers from low inter- and intra-rater reliability. Therefore, an automated sleep state classification method that operates on spatiotemporal WFCI data is desired.NEW METHOD:A hybrid network architecture consisting of a convolutional neural network (CNN) to extract spatial features of image frames and a bidirectional long short-term memory network (BiLSTM) with attention mechanism to identify temporal dependencies among different time points was proposed to classify WFCI data into states of wakefulness, NREM and REM sleep.RESULTS:Sleep states were classified with an accuracy of 84% and Cohen's kappa of 0.64. Gradient-weighted class activation maps revealed that the frontal region of the cortex carries more importance when classifying WFCI data into NREM sleep while posterior area contributes most to the identification of wakefulness. The attention scores indicated that the proposed network focuses on short- and long-range temporal dependency in a state-specific manner.COMPARISON WITH EXISTING METHOD:On a 3-hour WFCI recording, the CNN-BiLSTM achieved a kappa of 0.67, comparable to a kappa of 0.65 corresponding to the human EEG/EMG-based scoring.CONCLUSIONS:The CNN-BiLSTM effectively classifies sleep states from spatiotemporal WFCI data and will enable broader application of WFCI in sleep.

Wide-field calcium imaging (WFCI) that records neural calcium dynamics allows for identification of functional brain networks (FBNs) in mice that express genetically encoded calcium indicators. Estimating FBNs from WFCI data is commonly achieved by use of seed-based correlation (SBC) analysis and independent component analysis (ICA). These two methods are conceptually distinct and each possesses limitations. Recent success of unsupervised representation learning in neuroimage analysis motivates the investigation of such methods to identify FBNs. In this work, a novel approach referred as LSTM-AER, is proposed in which a long short-term memory (LSTM) autoencoder (AE) is employed to learn spatial-temporal latent embeddings from WFCI data, followed by an ordinary least square regression (R) to estimate FBNs. The goal of this study is to elucidate and illustrate, qualitatively and quantitatively, the FBNs identified by use of the LSTM-AER method and compare them to those from traditional SBC and ICA. It was observed that spatial FBN maps produced from LSTM-AER resembled those derived by SBC and ICA while better accounting for intra-subject variation, data from a single hemisphere, shorter epoch lengths and tunable number of latent components. The results demonstrate the potential of unsupervised deep learning-based approaches to identifying and mapping FBNs.

Despite progress in the design of protein binding proteins, the shape matching of binder to target has not yet reached that of highly evolved native protein-protein complexes, and previous design efforts have failed for hard targets such as the TNF receptor (TNFR1) that have relatively flat and polar surfaces. We reasoned that free diffusion starting from random noise could enable generation of extensive shape-matching binders to challenging targets, and tested this approach on TNFR1 and related super family members. The diffused TNFR1 binders have nanomolar affinities that increase to single-digit picomolar upon refinement by partial diffusion, and their specificities can be completely switched by partial diffusion in the context of other family members. The designs function as antagonists as monomers, and as superagonists when presented trivalently for OX40 and at higher valency for 4-1BB. The ability to design high -affinity and specific antagonists and agonists for a difficult but pharmacologically important class of proteins entirely in silico, without any large-scale screening or experimental optimization, presages a new era in which binders are made by computation rather than much more laborious and less controllable random screening approaches.

Measurements of gene expression or signal transduction activity are conventionally performed using methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm in which such biological activities are stably recorded to the genome. Enhancer-driven genomic recording of transcriptional activity in multiplex (ENGRAM) is based on the signal-dependent production of prime editing guide RNAs that mediate the insertion of signal-specific barcodes (symbols) into a genomically encoded recording unit. We show how this strategy can be used for multiplex recording of the cell-type-specific activities of dozens to hundreds of cis-regulatory elements with high fidelity, sensitivity and reproducibility. Leveraging signal transduction pathway-responsive cis-regulatory elements, we also demonstrate time- and concentration-dependent genomic recording of WNT, NF-κB and Tet-On activities. By coupling ENGRAM to sequential genome editing via DNA Typewriter1, we stably record information about the temporal dynamics of two orthogonal signalling pathways to genomic DNA. Finally we apply ENGRAM to integratively record the transient activity of nearly 100 transcription factor consensus motifs across daily windows spanning the differentiation of mouse embryonic stem cells into gastruloids, an in vitro model of early mammalian development. Although these are proof-of-concept experiments and much work remains to fully realize the possibilities, the symbolic recording of biological signals or states within cells, to the genome and over time, has broad potential to complement contemporary paradigms for how we make measurements in biological systems.