Biology subjects

Church, G. M.

Publications and source records attributed to Church, G. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Architectural fragility of gene regulatory networks underlies hematopoietic stem cell aging

Hematopoietic stem and progenitor cell (HSPC) aging contributes to immune dysfunction and age-associated disease, but its regulatory mechanisms remain unclear. Here, we present the largest single-cell multiome atlas of human circulating HSPCs to date, with >380,000 paired RNA and ATAC profiles across 77 donors. Beyond recapitulating established hallmarks of HSPC aging, we reconstructed a high-resolution gene regulatory network and identified a global rewiring in which stress-response and myeloid transcription factor (TF) programs expand, while self-renewal and lymphoid lineage-defining circuitry collapses. We associate these phenotypes with increased cis-regulatory entropy, including elevated transcriptomic noise, weakened peak-to-gene coupling, and chromatin peak broadening. This rewiring is selective, where TFs with GC-rich, promoter-proximal architectures are preserved or amplified, and complex, distal enhancer-dependent identity networks are eroded. Thus, these findings suggest that progressive entropic destabilization of gene regulatory architecture simultaneously drives stress hyperactivation, myeloid bias, and identity loss in aging HSPCs.

genomics↗

bioRxiv2026

Short single-stranded DNA oligonucleotides enable intracellular transcription of functional RNAs in mammalian cells

Programmable intracellular production of short functional RNAs underlies diverse applications ranging from gene regulation to genome engineering, but is often constrained by cloning workflows, RNA synthesis, or viral-vector delivery. Here, we introduce a single-stranded DNA (ssDNA)-driven intracellular transcription strategy in which short ssDNA templates function as transcriptional substrates when paired with orthogonal bacteriophage RNA polymerases (RNAPs). By embedding phage promoters within short hairpin structures to create a locally double-stranded recognition site, we enable robust RNAP-dependent transcription from ssDNA in mammalian cells. We demonstrate that ssDNA templates can drive production of functional CRISPR guides, supporting adenine base editing at reporter and endogenous loci and enabling CRISPR-based transcriptional activation across distinct human cell lines. These results establish ssDNA templates as compact, synthetically accessible inputs for RNA production in mammalian cells, with broad utility in synthetic biology and genome engineering.

bioengineering↗

bioRxiv2026

Genetic code expansion enables programmable covalent protein design

Covalent chemistry has transformed small-molecule drug discovery, yet analogous strategies for proteins remain largely inaccessible because covalent warheads cannot be readily integrated into biologics. Conventional genetic code expansion requires engineering a dedicated aminoacyl-tRNA synthetase for each new amino acid, rendering broad warhead screening impractical. Here we introduce AminoX, a platform that bypasses this limitation through direct tRNA acylation, enabling site-specific incorporation of chemically diverse non-standard amino acids (nsAAs), including covalent warhead nsAAs compatible with scalable biologic manufacturing and multifunctional nsAAs. Using a pooled mRNA display workflow, we screened more than 2,000 warhead-position combinations in machine learning-designed de novo miniproteins targeting CTLA-4, enabling parallel interrogation of covalent chemistry, linker geometry, and incorporation site. We confirmed covalent engagement on cells together with enhanced functional blockade. Finally, we demonstrate multifunctional nsAAs that combine covalent warheads with fluorogenic reporters for real-time detection of target engagement, as well as dual nsAA incorporation for macrocyclization and fluorescent imaging of covalent binding on cell surfaces. By uniting synthetic biology, chemical biology, generative protein design, and high-throughput functional selection, AminoX compresses covalent protein engineering timelines by orders of magnitude, accelerating the development of next-generation therapeutics, biosensors, and chemical probes.

synthetic biology↗

bioRxiv2026

DNA cut-ligation cyclization surpasses J-Factor limit by order of magnitude

We demonstrate DNA circularization efficiencies more than threefold greater than those predicted by classical Jacobson-Stockmayer theory for the ligation of linear double-stranded DNA. To quantify deviations from classical expectations, we experimentally calibrated the J-factor by ligating 452 bp DNA fragments bearing pre-cut, purified 4-nt overhangs at the optimal shortest minicircle length and at the highest DNA concentration; this reference value then enabled accurate calculation of expected cyclization efficiencies across the full range of DNA lengths (452-952 bp) and the three concentrations examined. These elevated cyclization efficiencies are enabled by simultaneous restriction cutting and ligation using the Type IIS enzyme BsaI-HFv2 in combination with T4 DNA ligase under finely optimized buffer conditions, yielding a 3.4-fold improvement over the classical estimate and achieving 75% cyclization at a high, practically relevant DNA concentration of 120 ng/{micro}l. We also identified a second enzyme, Esp3I, that exceeds the classical expectations by 2.3-fold (though less efficiently than BsaI-HFv2), while BbsI cut-ligation systematically underperformed expectations, providing insight into possible mechanisms underlying the outstanding performance of the first two enzymes. These results reveal a biologically mediated exception that overcomes the long-standing mechanistic expectations of Jacobson- Stockmayer theory and highlight the value of systematically screening enzyme combinations to discover additional systems capable of highly efficient intramolecular ligation.

molecular biology↗

bioRxiv2025

10,239 whole genomes with multiomic and clinical health information as the Korean population multiomic reference dataset

We present Korea10K, the largest genomic dataset of the Korean population, comprising 10,239 high-coverage whole genomes (mean depth 30x) with matched multiomic profiles and phenotype data. Korea10K achieves complete and near-complete discovery of very rare and ultra-rare alleles, respectively, at 9,000 Korean genomes. This dataset provides the high-quality population-specific imputation panel, enabling accurate inference of low-frequency variants. Admixture analyses confirm the genetic homogeneity of the Korean population, despite its diverse Y-chromosomal, mitochondrial, and HLA repertoires. This pattern reflects a long and continuous lineage history characterized by persistent internal admixture and genomic homogenization over thousands of years on the Korean peninsula. We also identified 16.8 million genomic variants that directly modify CG sites by creating or abolishing CG dinucleotides, providing the population-scale evidence of coordinated genomic-epigenomic regulatory mechanism in Koreans.

genomics↗

bioRxiv2025

Sourdough starter-inspired living materials grown from probiotic consortia with engineered division-of-labor

Genetic circuits enable precise control over cellular behavior. As circuits become more complex, implementing them within a single cell population becomes difficult. Tasks can instead be distributed in engineered consortia. However, selecting appropriate consortia and harnessing the natural traits of each species for such a division of labor remain unsolved challenges. Here, we report the creation of SINERGY, an engineered synthetic sourdough starter in which functional modules are distributed between the two constituent species. These modules, consisting of sensing, communication, and response, enable in vitro biosensing and in vivo drug delivery and modulate the gut ecosystem in an animal disease model. SINERGY not only preserves the safety and health-promoting properties of the natural components but also endows microbes with programmable functionalities, facilitating broad applications in biomedicine.

synthetic biology↗

bioRxiv2025

Combinatorial protein barcodes enable self-correcting neuron tracing with nanoscale molecular context

Mapping nanoscale neuronal morphology with molecular annotations is critical for understanding healthy and dysfunctional brain circuits. Current methods are constrained by image segmentation errors and by sample defects (e.g., signal gaps, section loss). Genetic strategies promise to overcome these challenges by using easily distinguishable cell identity labels. However, multicolor approaches are spectrally limited in diversity, whereas nucleic acid barcoding lacks a cellfilling morphology signal for segmentation. Here, we introduce PRISM (Protein-barcode Reconstruction via Iterative Staining with Molecular annotations), a platform that integrates combinatorial delivery of antigenically distinct, cell-filling proteins with tissue expansion, multi-cycle imaging, barcode-augmented reconstruction, and molecular annotation. Protein barcodes increase label diversity by >750-fold over multicolor labeling and enable morphology reconstruction with intrinsic error correction. We acquired a [~]10 million {micro}m3 volume of mouse hippocampal area CA2/3, multiplexed across 23 barcode antigen and synaptic marker channels. By combining barcodes with shape information, we achieve an 8x increase in automatic tracing accuracy of genetically labelled neurons. We demonstrate PRISM supports automatic proofreading across micron-scale spatial gaps and reconnects neurites across discontinuities spanning hundreds of microns. Using PRISMs molecular annotation capability, we map the distribution of synapses onto traced neural morphology, characterizing challenging synaptic structures such as thorny excrescences (TEs), and discovering a size correlation among spatially proximal TEs on the same dendrite. PRISM thus supports selfcorrecting neuron reconstruction with molecular context.

neuroscience↗

bioRxiv2025

An Expanded Repertoire of tRNA Sources for Cell-Free Protein Synthesis

Cell-free expression systems (CFE) are flexible protein translation platforms that simplify the central dogma into an accessible reaction space. Within these systems, bulk transfer RNAs (tRNAs) are critical substrates which deliver amino acids to the elongating ribosome. For years, CFE systems were completed with commercially available tRNA isolated from E. coli MRE600. All commercial sources of tRNA have since been discontinued, jeopardizing future work in all applications of cell-free translation. Here, we address this need by repurposing previously described tRNA isolation methods to produce tRNAs suitable for CFE applications. We isolated the tRNA pools of E. coli strains A19, BL21(DE3), and Rosetta2 BL21(DE3), finding A19 tRNAs but not BL21(DE3) or Rosetta2 BL21(DE3) capable of robust in vitro translation. We determined the abundances of individual tRNAs using tRNA-seq, finding BL21(DE3) and Rosetta2 BL21(DE3) contained outsized abundances of several tRNAs, compromising translation activity. Using codon optimization strategies which align codon usage to tRNA abundance, we were able to mitigate the impact of misaligned tRNA abundances. We extended these studies to V. natriegens, a promising platform for synthetic biology and CFE. We find that neither exogenous V. natriegens tRNAs nor codon optimization are viable options to improve translation yields. Our work here highlights the importance of tRNA abundance within the context of CFE, and simultaneously addresses a critical challenge within cell-free translation.

biochemistry↗

bioRxiv2025

Simulating a for-loop in the human genome: Design and evaluation of recombinase genetic programs that count to three.

Pluripotent cells specialize into numerous cell types by receiving external signals, making fate decisions, and executing differentiation functions - a paradigm similar to computer algorithms. While advances in biosensor design have enabled cells to respond to diverse stimuli, the ability to maintain a synthetic memory of the cells experiences that then informs its behaviors remains elusive. Here, we developed a system for cellular memory-driven behaviors by simulating a "for-loop" that counts to three using recombinase STepwise gene Expression Programs (STEPs). STEPs were genomically integrated into human cells, genotyped through targeted nanopore sequencing, and evaluated for function through changes in fluorescent reporter expression. While all STEPs were capable of heritable memory and sequential gene expression, the STEP design using tyrosine recombinases for successive excisions (TRex) significantly outperformed the others tested. We then used live cell imaging to track TRex cells as they incremented from count zero to three and observed the successive emergence of four cell states from an initially homogeneous population. The STEPs framework provides biological memory and conditional expression capabilities that, coupled with input mechanisms such as biosensors, can start to approach a programming language for biology.

synthetic biology↗

bioRxiv2025

A simple, ultrastable, and cost-effective oxygen-scavenging system for long-term DNA-PAINT imaging

DNA-PAINT (Points Accumulation in Nanoscale Topography) is a super-resolution microscopy technique capable of nanoscale imaging through the transient binding of fluorescently labeled imager strands to complementary DNA docking strands. Imager strands can be continuously replenished from an effectively infinite pool, making DNA-PAINT inherently resistant to photobleaching. However, extended DNA-PAINT imaging is limited by the formation of reactive oxygen species (ROS), which damage docking strands and reduce localization sampling over time. Although the state-of-the-art oxygen-scavenging system (OSS) can mitigate this damage, its enzymatic components degrade over time, reducing its performance, robustness and utility. Here, we introduce a simple, enzyme-free oxygen scavenging buffer based on sodium sulfite (Na2SO3) that overcomes these challenges. Our optimized formulation, combining Na2SO3 with Trolox (SST), effectively preserves docking strand integrity for over 24 hours and enhances long-term imaging performance. SST improves buffer stability tenfold, is easy to prepare and reduces costs by more than 90%, providing a robust, cost-effective, high-performance OSS for extended DNA-PAINT imaging.

biophysics↗

bioRxiv2025

Altered Protein Phosphorylation in a Novel Midbrain Organoid Model for Bipolar Disorder

Bipolar disorder (BD) is a severe psychiatric condition marked by episodes of mania and depression, with neurotransmitter imbalance in the midbrain believed to play a critical role in its pathophysiology. Despite this, there is currently no validated midbrain model for examining BD-associated molecular changes available. Leveraging recent advances in stem cell technology, we developed a midbrain organoid model using human induced pluripotent stem cells (hiPSCs) from BD patients and healthy controls (CTR). To address issues of variability and enhance the throughput in organoid production, we implemented liquid handling and high-content imaging techniques. Quality control metrics were established to identify organoids unsuitable for further study. Electrophysiological analysis via high-density microelectrode arrays (MEAs) revealed significantly elevated neuronal properties in individual BD organoids, including increased mean amplitude, conduction velocity, and extended axonal and dendritic growth. Transcriptome and proteome analyses indicated significant dysregulation of BD-relevant signaling pathways--such as those involving phosphatidylinositol, glycogen synthase kinase-3 beta, and AKT. Notably, we identified dysregulated casein kinase 2 (CSNK2A1) and calmodulin 3 (CALM3) in BD organoids, which were reversed by lithium treatment, highlighting potential novel targets for therapeutic intervention. This study validates the midbrain organoid model as a valuable tool for exploring the molecular underpinnings of BD and identifying new treatment avenues.

cell biology↗

bioRxiv2025

Multiplex-edited mice recapitulate woolly mammoth hair phenotypes

The woolly mammoth (Mammuthus primigenius) possessed a thick woolly coat and other cold-adaptive traits that enabled survival in harsh arctic environments. Current de-extinction efforts focus on genetically modifying the closely related Asian elephant to express woolly mammoth traits. In this study, we establish a multiplex-edited mouse model with modifications in genes associated with hair morphology and lipid metabolism, enabling insights into traits involved in developing woolly hair textures. Our optimized workflows achieved high editing efficiencies and produced genetically modified mice with simultaneous editing of up to seven different genes. Selected modifications include loss-of-function mutations in Fgf5, Tgm3, and Fam83g, among others. The resulting mice display exaggerated hair phenotypes including curly, textured coats, and golden-brown hair. This study advances methods of rapid establishment of complex genetic models. These approaches inform de-extinction efforts and research involving the genetic basis of mammalian hair development.

genetics↗

bioRxiv2025

Engineering gene drive docking sites in a haplolethal locus in Anopheles gambiae

Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing permits the cell to survive. Here we outline the development and characterization of two {Phi}C31-Recombination mediated cassette exchange (RMCE) gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants that reveal a major hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for future development of gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.

synthetic biology↗

bioRxiv2025

DNA-PAINT imaging with hydrogel imprinting and clearing

Hydrogel-embedding is a versatile technique in fluorescence microscopy, offering stabilization, optical clearing, and the physical expansion of biological specimens. DNA-PAINT is a super-resolution microscopy approach based on the diffusion and transient binding of fluorescently labeled oligos, but its feasibility in hydrogels has not yet been explored. In this study, we demonstrate that polyacrylamide hydrogels support sufficient diffusion for effective DNA-PAINT imaging. Using acrydite-anchored oligonucleotides imprinted from patterned DNA origami nanostructures and microtubule filaments in fixed cells, we find that hydrogel embedding preserves docking strand positioning at the nanoscale. Sample clearing via protease treatment had minor structural effects on the microtubule structure, and enhanced diffusion and accessibility to hydrogel-imprinted docking strands. Our work demonstrates promising potential for diffusion and binding-based fluorescence imaging applications in hydrogel-embedded samples. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/638341v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@e508c7org.highwire.dtl.DTLVardef@a36a4eorg.highwire.dtl.DTLVardef@985e9aorg.highwire.dtl.DTLVardef@16afff4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Synthetic ratio computation for programming population composition and multicellular morphology

Recent advancements in genetic engineering have provided diverse tools for artificially synthesizing population diversity in both prokaryotic and eukaryotic systems. However, achieving precise control over the ratios of multiple cell types within a population derived from a single founder remains a significant challenge. In this study, we introduce a suite of recombinase-mediated genetic devices designed to achieve accurate population ratio control, enabling the distribution of distinct functionalities across multiple cell types. We systematically evaluated key parameters influencing recombination efficiency and developed data-driven models to reliably predict binary differentiation outcomes. Using these devices, we implemented parallel and series circuit topologies to create user-defined, complex cell fate branching programs. These branching devices facilitated the autonomous differentiation of precision fermentation consortia from a single founder strain, optimizing cell-type ratios for applications such as pigmentation and cellulose degradation. Beyond biomanufacturing, we engineered multicellular aggregates with genetically encoded morphologies by coordinating self-organization through cell adhesion molecules (CAMs). Our work provides a comprehensive characterization of recombinase-based cell fate branching mechanisms and introduces a novel approach for the bottom-up, high-resolution construction of synthetic consortia and multicellular assemblies.

synthetic biology↗

bioRxiv2024

Site of breast cancer metastasis is independent of single nutrient levels

Cancer metastasis is a major contributor to patient morbidity and mortality1, yet the factors that determine the organs where cancers can metastasize are incompletely understood. In this study, we quantify the absolute levels of over 100 nutrients available across multiple tissues in mice and investigate how this relates to the ability of breast cancer cells to grow in different organs. We engineered breast cancer cells with broad metastatic potential to be auxotrophic for specific nutrients and assessed their ability to colonize different organs. We then asked how tumor growth in different tissues relates to nutrient availability and tumor biosynthetic activity. We find that single nutrients alone do not define the sites where breast cancer cells can grow as metastases. Additionally, we identify purine synthesis as a requirement for tumor growth and metastasis across many tissues and find that this phenotype is independent of tissue nucleotide availability or tumor de novo nucleotide synthesis activity. These data suggest that a complex interplay of multiple nutrients within the microenvironment dictates potential sites of metastatic cancer growth, and highlights the interdependence between extrinsic environmental factors and intrinsic cellular properties in influencing where breast cancer cells can grow as metastases.

cancer biology↗

bioRxiv2024

Expansion in situ genome sequencing links nuclear abnormalities to hotspots of aberrant euchromatin repression

Microscopy and genomics are both used to characterize cell function, but approaches to connect the two types of information are lacking, particularly at subnuclear resolution. While emerging multiplexed imaging methods can simultaneously localize genomic regions and nuclear proteins, their ability to accurately measure DNA-protein interactions is constrained by the diffraction limit of optical microscopy. Here, we describe expansion in situ genome sequencing (ExIGS), a technology that enables sequencing of genomic DNA and superresolution localization of nuclear proteins in single cells. We applied ExIGS to fibroblast cells derived from an individual with Hutchinson-Gilford progeria syndrome to characterize how variation in nuclear morphology affects spatial chromatin organization. Using this data, we discovered that lamin abnormalities are linked to hotspots of aberrant euchromatin repression that may erode cell identity. Further, we show that lamin abnormalities heterogeneously increase the repressive environment of the nucleus in tissues and aged cells. These results demonstrate that ExIGS may serve as a generalizable platform for connecting nuclear abnormalities to changes in gene regulation across disease contexts.

genomics↗

bioRxiv2024

Manufacturing-Aware Generative Model Architectures Enable Biological Sequence Design and Synthesis at Petascale

We introduce a method to reduce the cost of synthesizing proteins and other biological sequences designed by a generative model by as much as a trillion-fold. In particular, we make our generative models manufacturing-aware, such that model-designed sequences can be efficiently synthesized in the real world with extreme parallelism. We demonstrate by training and synthesizing samples from generative models of antibodies, T cell antigens and DNA polymerases. For example, we train a manufacturing-aware generative model on 300 million observed human antibodies and synthesize[~] 1017 generated designs from the model, achieving a sample quality comparable to a state-of-the-art protein language model, at a cost of 103 dollars. Using previous methods, synthesis of a library of the same accuracy and size would cost roughly a quadrillion (1015) dollars.

bioengineering↗