Xiaojing Gao

All Publications

• Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems. bioRxiv : the preprint server for biology Katz, N., An, C., Lee, Y. J., Tycko, J., Zhang, M., Kang, J., Bintu, L., Bassik, M. C., Huang, W. H., Gao, X. J. 2024

  Abstract

  Gene therapy holds great therapeutic potential. Yet, controlling cargo expression in single cells is limited due to the variability of delivery methods. We implement an incoherent feedforward loop based on proteolytic cleavage of CRISPR-Cas activation or inhibition systems to reduce gene expression variability against the variability of vector delivery. We demonstrate dosage control for activation and inhibition, post-delivery tuning, and RNA-based delivery, for a genome-integrated marker. We then target the RAI1 gene, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes, Smith-Magenis-Syndrome (SMS) and Potocki-Lupski-Syndrome, respectively. We demonstrate dosage control for RAI1 activation in HEK293s, Neuro-2As, and mouse cortical neurons via AAVs and lentiviruses. Finally, we activate the intact RAI1 copy in SMS patient-derived cells to an estimated two-copy healthy range, avoiding the harmful three-copy regime. Our circuit paves the way for viable therapy in dosage-sensitive disorders, creating precise and tunable gene regulation systems for basic and translational research.

  View details for DOI 10.1101/2024.10.09.617463

  View details for PubMedID 39416069

  View details for PubMedCentralID PMC11482798

• Post-Transcriptional Modular Synthetic Receptors. bioRxiv : the preprint server for biology Zhang, X., Mille-Fragoso, L. S., Kaseniit, K. E., Call, C. C., Zhang, M., Hu, Y., Xie, Y., Gao, X. J. 2024

  Abstract

  Inspired by the power of transcriptional synthetic receptors and hoping to complement them to expand the toolbox for cell engineering, we establish LIDAR (Ligand-Induced Dimerization Activating RNA editing), a modular post-transcriptional synthetic receptor platform that harnesses RNA editing by ADAR. LIDAR is compatible with various receptor architectures in different cellular contexts, and enables the sensing of diverse ligands and the production of functional outputs. Furthermore, LIDAR can sense orthogonal signals in the same cell and produce synthetic spatial patterns, potentially enabling the programming of complex multicellular behaviors. Finally, LIDAR is compatible with compact encoding and can be delivered by synthetic mRNA. Thus, LIDAR expands the family of synthetic receptors, holding the promise to empower basic research and therapeutic applications.

  View details for DOI 10.1101/2024.05.03.592453

  View details for PubMedID 38746461

  View details for PubMedCentralID PMC11092781

• Compact Programmable Control of Protein Secretion in Mammalian Cells. bioRxiv : the preprint server for biology Vlahos, A. E., Call, C. C., Kadaba, S. E., Guo, S., Gao, X. J. 2023

  Abstract

  Synthetic biology currently holds immense potential to engineer the spatiotemporal control of intercellular signals for biomedicine. Programming behaviors using protein-based circuits has advantages over traditional gene circuits such as compact delivery and direct interactions with signaling proteins. Previously, we described a generalizable platform called RELEASE to enable the control of intercellular signaling through the proteolytic removal of ER-retention motifs compatible with pre-existing protease-based circuits. However, these tools lacked the ability to reliably program complex expression profiles and required numerous proteases, limiting delivery options. Here, we harness the recruitment and antagonistic behavior of endogenous 14-3-3 proteins to create RELEASE-NOT to turn off protein secretion in response to protease activity. By combining RELEASE and RELEASE-NOT, we establish a suite of protein-level processing and output modules called Compact RELEASE (compRELEASE). This innovation enables functions such as logic processing and analog signal filtering using a single input protease. Furthermore, we demonstrate the compactness of the post-translational design by using polycistronic single transcripts to engineer cells to control protein secretion via lentiviral integration and leverage mRNA delivery to selectively express cell surface proteins only in engineered cells harboring inducible proteases. CompRELEASE enables complex control of protein secretion and enhances the potential of synthetic protein circuits for therapeutic applications, while minimizing the overall genetic payload.

  View details for DOI 10.1101/2023.10.04.560774

  View details for PubMedID 37873144

• Modular, programmable RNA sensing using ADAR editing in living cells. Nature biotechnology Kaseniit, K. E., Katz, N., Kolber, N. S., Call, C. C., Wengier, D. L., Cody, W. B., Sattely, E. S., Gao, X. J. 2022

  Abstract

  With the increasing availability of single-cell transcriptomes, RNA signatures offer a promising basis for targeting living cells. Molecular RNA sensors would enable the study of and therapeutic interventions for specific cell types/states in diverse contexts, particularly in human patients and non-model organisms. Here we describe a modular, programmable system for live RNA sensing using adenosine deaminases acting on RNA (RADAR). We validate, and then expand, our basic design, characterize its performance, and analyze its compatibility with human and mouse transcriptomes. We identify strategies to boost output levels and improve the dynamic range. Additionally, we show that RADAR enables compact AND logic. In addition to responding to transcript levels, RADAR can distinguish disease-relevant sequence alterations of transcript identities, such as point mutations and fusions. Finally, we demonstrate that RADAR is a self-contained system with the potential to function in diverse organisms.

  View details for DOI 10.1038/s41587-022-01493-x

  View details for PubMedID 36198772

• Protease-controlled secretion and display of intercellular signals. Nature communications Vlahos, A. E., Kang, J., Aldrete, C. A., Zhu, R., Chong, L. S., Elowitz, M. B., Gao, X. J. 2022; 13 (1): 912

  Abstract

  To program intercellular communication for biomedicine, it is crucial to regulate the secretion and surface display of signaling proteins. If such regulations are at the protein level, there are additional advantages, including compact delivery and direct interactions with endogenous signaling pathways. Here we create a modular, generalizable design called Retained Endoplasmic Cleavable Secretion (RELEASE), with engineered proteins retained in the endoplasmic reticulum and displayed/secreted in response to specific proteases. The design allows functional regulation of multiple synthetic and natural proteins by synthetic protease circuits to realize diverse signal processing capabilities, including logic operation and threshold tuning. By linking RELEASE to additional sensing and processing circuits, we can achieve elevated protein secretion in response to "undruggable" oncogene KRAS mutants. RELEASE should enable the local, programmable delivery of intercellular cues for a broad variety of fields such as neurobiology, cancer immunotherapy and cell transplantation.

  View details for DOI 10.1038/s41467-022-28623-y

  View details for PubMedID 35177637

• Engineering multiple levels of specificity in an RNA viral vector BioRxiv Gao, X. J., Chong, L. S., Ince, M. H., Kim, M. S., Elowitz, M. B. 2020

  View details for DOI 10.1101/2020.05.27.119909

• Programmable protein circuits in living cells. Science (New York, N.Y.) Gao, X. J., Chong, L. S., Kim, M. S., Elowitz, M. B. 2018; 361 (6408): 1252-1258

  Abstract

  Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnological applications.

  View details for DOI 10.1126/science.aat5062

  View details for PubMedID 30237357

  View details for PubMedCentralID PMC7176481

• Perspectives on synthetic protein circuits in mammalian cells CURRENT OPINION IN BIOMEDICAL ENGINEERING Aldrete, C. A., An, C., Call, C. C., Gao, X. J., Vlahos, A. E. 2024; 32

  View details for DOI 10.1016/j.cobme.2024.100555

  View details for Web of Science ID 001308751200001

• Perspectives on Synthetic Protein Circuits in Mammalian Cells. Current opinion in biomedical engineering Aldrete, C. A., An, C., Call, C. C., Gao, X. J., Vlahos, A. E. 2024; 32

  Abstract

  Mammalian synthetic biology aims to engineer cellular behaviors for therapeutic applications, such as enhancing immune cell efficacy against cancers or improving cell transplantation outcomes. Programming complex biological functions necessitates an understanding of molecular mechanisms governing cellular responses to stimuli. Traditionally, synthetic biology has focused on transcriptional circuits, but recent advances have led to the development of synthetic protein circuits, leveraging programmable binding, proteolysis, or phosphorylation to modulate protein interactions and cellular functions. These circuits offer advantages including robust performance, rapid functionality, and compact design, making them suitable for cellular engineering or gene therapies. This review outlines the post-translational toolkit, emphasizing synthetic protein components utilizing proteolysis or phosphorylation to program mammalian cell behaviors. Finally, we focus on key differences between rewiring native signaling pathways and creating orthogonal behaviors, alongside a proposed framework for translating synthetic protein circuits from tool development to pre-clinical applications in biomedicine.

  View details for DOI 10.1016/j.cobme.2024.100555

  View details for PubMedID 39372446

  View details for PubMedCentralID PMC11448451

• Protease-Driven Phase Separation of Elastin-Like Polypeptides. Biomacromolecules Wirtz, B. M., Yun, A. G., Wick, C., Gao, X. J., Mai, D. J. 2024

  Abstract

  Elastin-like polypeptides (ELPs) are a promising material platform for engineering stimuli-responsive biomaterials, as ELPs undergo phase separation above a tunable transition temperature. ELPs with phase behavior that is isothermally regulated by biological stimuli remain attractive for applications in biological systems. Herein, we report protease-driven phase separation of ELPs. Protease-responsive "cleavable" ELPs comprise a hydrophobic ELP block connected to a hydrophilic ELP block by a protease cleavage site linker. The hydrophilic ELP block acts as a solubility tag for the hydrophobic ELP block, creating a temperature window in which the cleavable ELP reactant is soluble and the proteolytically generated hydrophobic ELP block is insoluble. Within this temperature window, isothermal, protease-driven phase separation occurs when a critical concentration of hydrophobic cleavage product accumulates. Furthermore, protease-driven phase separation is generalizable to four compatible protease-cleavable ELP pairings. This work presents exciting opportunities to regulate ELP phase behavior in biological systems using proteases.

  View details for DOI 10.1021/acs.biomac.4c00346

  View details for PubMedID 38980747

• Topological Organization of Ventral Tegmental Area Connectivity Revealed by Viral-Genetic Dissection of Input-Output Relations. Cell reports Beier, K. T., Gao, X. J., Xie, S., DeLoach, K. E., Malenka, R. C., Luo, L. 2019; 26 (1): 159

  Abstract

  Viral-genetic tracing techniques have enabled mesoscale mapping of neuronal connectivity by teasing apart inputs to defined neuronal populations in regions with heterogeneous cell types. We previously observed input biases to output-defined ventral tegmental area dopamine (VTA-DA) neurons. Here, we further dissect connectivity in the VTA by defining input-output relations of neurochemically and output-defined neuronal populations. By expanding our analysis to include input patterns to subtypes of excitatory (vGluT2-expressing) or inhibitory (GAD2-expressing) populations, we find that the output site, rather than neurochemical phenotype, correlates with whole-brain inputs of each subpopulation. Lastly, we find that biases in input maps to different VTA neurons can be generated using publicly available whole-brain output mapping datasets. Our comprehensive dataset and detailed spatial analysis suggest that connection specificity in the VTA is largely a function of the spatial location of the cells within the VTA.

  View details for PubMedID 30605672

• Synthetic biology: Precision timing in a cell. Nature Gao, X. J., Elowitz, M. B. 2016; 538 (7626): 462-463

  View details for DOI 10.1038/nature19478

  View details for PubMedID 27732579

• Cas9-triggered chain ablation of cas9 as a gene drive brake. Nature biotechnology Wu, B. n., Luo, L. n., Gao, X. J. 2016; 34 (2): 137–38

  View details for PubMedID 26849513

• Viral-genetic tracing of the input-output organization of a central noradrenaline circuit. Nature Schwarz, L. A., Miyamichi, K., Gao, X. J., Beier, K. T., Weissbourd, B., DeLoach, K. E., Ren, J., Ibanes, S., Malenka, R. C., Kremer, E. J., Luo, L. 2015; 524 (7563): 88-92

  Abstract

  Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input-output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The 'tracing the relationship between input and output' method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input-output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

  View details for DOI 10.1038/nature14600

  View details for PubMedID 26131933

• Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping CELL Beier, K. T., Steinberg, E. E., DeLoach, K. E., Xie, S., Miyamichi, K., Schwarz, L., Gao, X. J., Kremer, E. J., Malenka, R. C., Luo, L. 2015; 162 (3): 622-634

  Abstract

  Dopamine (DA) neurons in the midbrain ventral tegmental area (VTA) integrate complex inputs to encode multiple signals that influence motivated behaviors via diverse projections. Here, we combine axon-initiated viral transduction with rabies-mediated trans-synaptic tracing and Cre-based cell-type-specific targeting to systematically map input-output relationships of VTA-DA neurons. We found that VTA-DA (and VTA-GABA) neurons receive excitatory, inhibitory, and modulatory input from diverse sources. VTA-DA neurons projecting to different forebrain regions exhibit specific biases in their input selection. VTA-DA neurons projecting to lateral and medial nucleus accumbens innervate largely non-overlapping striatal targets, with the latter also sending extensive extra-striatal axon collaterals. Using electrophysiology and behavior, we validated new circuits identified in our tracing studies, including a previously unappreciated top-down reinforcing circuit from anterior cortex to lateral nucleus accumbens via VTA-DA neurons. This study highlights the utility of our viral-genetic tracing strategies to elucidate the complex neural substrates that underlie motivated behaviors.

  View details for DOI 10.1016/j.cell.2015.07.015

  View details for Web of Science ID 000358801800020

  View details for PubMedCentralID PMC4522312

• A transcriptional reporter of intracellular Ca(2+) in Drosophila. Nature neuroscience Gao, X. J., Riabinina, O., Li, J., Potter, C. J., Clandinin, T. R., Luo, L. 2015; 18 (6): 917-925

  Abstract

  Intracellular Ca(2+) is a widely used neuronal activity indicator. Here we describe a transcriptional reporter of intracellular Ca(2+) (TRIC) in Drosophila that uses a binary expression system to report Ca(2+)-dependent interactions between calmodulin and its target peptide. We found that in vitro assays predicted in vivo properties of TRIC and that TRIC signals in sensory systems depend on neuronal activity. TRIC was able to quantitatively monitor neuronal responses that changed slowly, such as those of neuropeptide F-expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis cells to food and arousal. Furthermore, TRIC-induced expression of a neuronal silencer in nutrient-activated cells enhanced stress resistance, providing a proof of principle that TRIC can be used for circuit manipulation. Thus, TRIC facilitates the monitoring and manipulation of neuronal activity, especially those reflecting slow changes in physiological states that are poorly captured by existing methods. TRIC's modular design should enable optimization and adaptation to other organisms.

  View details for DOI 10.1038/nn.4016

  View details for PubMedID 25961791

  View details for PubMedCentralID PMC4446202

• Extremely sparse olfactory inputs are sufficient to mediate innate aversion in Drosophila. PloS one Gao, X. J., Clandinin, T. R., Luo, L. 2015; 10 (4)

  Abstract

  Innate attraction and aversion to odorants are observed throughout the animal kingdom, but how olfactory circuits encode such valences is not well understood, despite extensive anatomical and functional knowledge. In Drosophila melanogaster, ~50 types of olfactory receptor neurons (ORNs) each express a unique receptor gene, and relay information to a cognate type of projection neurons (PNs). To examine the extent to which the population activity of ORNs is required for olfactory behavior, we developed a genetic strategy to block all ORN outputs, and then to restore output in specific types. Unlike attraction, aversion was unaffected by simultaneous silencing of many ORNs, and even single ORN types previously shown to convey neutral valence sufficed to mediate aversion. Thus, aversion may rely on specific activity patterns in individual ORNs rather than the number or identity of activated ORNs. ORN activity is relayed into the brain by downstream circuits, with excitatory PNs (ePN) representing a major output. We found that silencing the majority of ePNs did not affect aversion, even when ePNs directly downstream of single restored ORN types were silenced. Our data demonstrate the robustness of olfactory aversion, and suggest that its circuit mechanism is qualitatively different from attraction.

  View details for DOI 10.1371/journal.pone.0125986

  View details for PubMedID 25927233

  View details for PubMedCentralID PMC4416024

• Drosophila chemotaxis FLY Gao, X. J. 2014; 8 (1): 3-6

  Abstract

  Chemotaxis, the ability to direct movements according to chemical cues in the environment, is important for the survival of most organisms. In our original article, we combined a quantitative behavioral assay with genetic manipulations to dissect the neural substrate for chemotaxis. In this Extra View article, we offer a more chronological narration of the findings leading to our key conclusion that aversion engages specific motor-related circuits and kinematics. We speculate on the separation and crosstalk between aversion and attraction circuits in the brain and the ventral nerve cord, and the implication for valence encoding in the olfactory system.

  View details for DOI 10.4161/fly.26685

  View details for Web of Science ID 000332985000001

  View details for PubMedCentralID PMC3974891

• Specific Kinematics and Motor-Related Neurons for Aversive Chemotaxis in Drosophila CURRENT BIOLOGY Gao, X. J., Potter, C. J., Gohl, D. M., Silies, M., Katsov, A. Y., Clandinin, T. R., Luo, L. 2013; 23 (13): 1163-1172

  Abstract

  Chemotaxis, the ability to direct movements according to chemical cues in the environment, is important for the survival of most organisms. The vinegar fly, Drosophila melanogaster, displays robust olfactory aversion and attraction, but how these behaviors are executed via changes in locomotion remains poorly understood. In particular, it is not clear whether aversion and attraction bidirectionally modulate a shared circuit or recruit distinct circuits for execution.Using a quantitative behavioral assay, we determined that both aversive and attractive odorants modulate the initiation and direction of turns but display distinct kinematics. Using genetic tools to perturb these behaviors, we identified specific populations of neurons required for aversion, but not for attraction. Inactivation of these populations of cells affected the completion of aversive turns, but not their initiation. Optogenetic activation of the same populations of cells triggered a locomotion pattern resembling aversive turns. Perturbations in both the ellipsoid body and the ventral nerve cord, two regions involved in motor control, resulted in defects in aversion.Aversive chemotaxis in vinegar flies triggers ethologically appropriate kinematics distinct from those of attractive chemotaxis and requires specific motor-related neurons.

  View details for DOI 10.1016/j.cub.2013.05.008

  View details for Web of Science ID 000321605600017

  View details for PubMedID 23770185

• A versatile in vivo system for directed dissection of gene expression patterns NATURE METHODS Gohl, D. M., Silies, M. A., Gao, X. J., Bhalerao, S., Luongo, F. J., Lin, C., Potter, C. J., Clandinin, T. R. 2011; 8 (3): 231-U71

  Abstract

  Tissue-specific gene expression using the upstream activating sequence (UAS)–GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we developed a convertible genetic platform, the integrase swappable in vivo targeting element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient in vivo manipulations, making it possible to generate many swaps in parallel. This system is modular, allowing future genetic tools to be easily incorporated into the existing framework.

  View details for DOI 10.1038/NMETH.1561

  View details for Web of Science ID 000287734800014

  View details for PubMedID 21473015

  View details for PubMedCentralID PMC3079545