Biology subjects

Huynh, N.

Publications and source records attributed to Huynh, N..

11 recordsLinked to original sources

bioRxiv2026

AI discovery of sequence rules for RNA polymerase II pausing revises the pause-release model of gene activation

The promoter-proximal pause (PPP), at most metazoan genes, is believed to prime transcriptional activation by release of paused RNAPII into the gene. We tested the pause-release model by abolishing the PPP and monitoring gene activation. An LLM-guided AI discovered that upstream G's and a G T/C dinucleotide robustly discriminates pauses from non-pauses. Insertion of G-less sequences into NDRG1 and HSP90AA1 abolished the PPP in an orientation-dependent way, but did not impair activation by hypoxia or heat-shock. Hence pause-release is dispensable for these responses. The 5' peak of RNAPII detected by ChIP persisted even when pausing, detected by NET-seq was absent. Instead of pausing, these promoter-proximal polymerases may undergo abortive initiation. RNAPII C-terminal domain (CTD) deletion suppresses the PPP and shifts it upstream without affecting sequence-dependent pausing, consistent with promoter-tethering by this IDR. We propose that PPP formation is initiated by G-dependent arrest, and stabilized by multiple factors including CTD-dependent tethering.

molecular biology↗

bioRxiv2026

Concordant transcriptional and morphological remodeling revealed by in vivo Perturb-CLEAR

The principle that form follows function has long guided thinking in biology and architecture alike. In the nervous system, however, form does more than reflect function: neuronal morphology actively constrains input patterns, synaptic integration, and circuit wiring. During postnatal neurodevelopment, dendritic architectures are assembled and remodeled through genetically encoded programs and activities, transforming molecular programs into circuit architecture. However, dendritic morphogenesis has been difficult to quantify at scale while systematically testing how genetic variants, including neurodevelopmental disorder (NDD) risk genes, alter these structures in vivo. We developed Perturb-CLEAR, which integrates pooled CRISPR screening and whole-mount imaging to quantify brain-wide cytoarchitecture, and paired it with Perturb-seq to link structural phenotypes to transcriptomic changes. Applying Perturb-CLEAR to the developing mouse cortex revealed morphogenesis trajectories accompanied by transcriptomic dynamics. Moreover, systematic perturbation of NDD risk genes uncovered gene-specific multimodal phenotypes. Adnp perturbation remodels basal dendrites in L4/5 IT (intratelencephalic) neurons but not other dendritic compartments or cell types, alongside consistent transcriptional shifts. Combined morphology and transcriptome analyses link NDD risk genes to concordant multimodal cellular phenotypes in the developing brain, highlighting diverse paths of perturbation effect propagation across modalities.

neuroscience↗

bioRxiv2026

Genome-scale functional mapping of the mammalian whole brain with in vivo Perturb-seq

Functional genomics studies have provided critical insights into cell type-specific gene regulatory programs, but to date most have been conducted in wild-type tissues or cell cultures. Here, we present a gene expression functional atlas across the mouse brain. We use an enhanced in vivo Perturb-seq platform to analyze transcriptome-wide responses to loss of 1,947 disease-associated genes, profiling over 7.7 million cells spanning major brain regions and neuronal populations. We find striking cell-type-specific essentiality and transcriptional programs and show that closely related disease genes such as two NMDA receptor subunits can drive opposing transcriptional programs. Together, this work reveals insights into the genetics and mechanisms of neurodevelopmental, psychiatric, and neurodegenerative diseases in vivo, paving the way for the design of future genetic medicine.

genomics↗

bioRxiv2026

In vivo multiomic Perturb-seq with enhanced nuclear gRNA capture

In vivo CRISPR screening with joint transcriptomic and chromatin readouts has been limited by inefficient recovery of gRNAs from nuclei. Here, we developed in vivo multiomic Perturb-seq, an effective platform combining nuclear transcript anchoring with gRNA-specific capture and amplification to enable high-fidelity, high-recovery gRNA assignment and scalable perturbation-resolved single-nucleus multiomics. Applying this platform to interrogate neurodevelopmental disorder risk genes in the developing cortex reveals cell-type-specific transcriptomic and epigenomic perturbation phenotypes.

genomics↗

bioRxiv2025

Integration of Multiomic and Multi-phenotypic Data Identifies Biological Pathways Associated with Physical Fitness

Unraveling the complex associations between human phenotypes and molecular pathways can pave the way to improved health and performance, but faces a fundamental challenge: the measurable genes, proteins, and metabolites vastly outnumber the participants in even the largest studies, yielding spurious correlations. To address this imbalance, we have developed a bioinformatic framework and computational approach ("PhenoMol") to discover biological drivers of phenotypic characteristics that integrates all available phenotypic data predictive of outcomes and reduces multi-omic data dimensionality by generating "expression circuits" via graph theory constrained by prior biological knowledge of molecular interactions. We applied PhenoMol to analyze causal patterns and predict elite physical performance in a healthy cohort with deep physiological, physical, behavioral, cognitive, and molecular characterization. PhenoMol outperforms regression models based on equivalent analytic methodologies that do not employ network biology for dimensionality reduction. The PhenoMol software is provided for future studies.

bioinformatics↗

bioRxiv2025

CellFIE: Integrating Pathway Discovery With Pooled Profiling of Perturbations Uncovers Pathways of Huntington's Disease, Including Genetic Modifiers of Neuronal Development and Morphology

Genomic screens and GWAS are powerful tools for identifying disease-modifying genes, but it is often challenging to understand the pathways by which these genes function. Here, we take an integrated approach that combines network analysis and an imaging-based pooled genetic perturbation study to examine modifiers of Huntingtons disease (HD). The computational analysis highlighted several genes in a subnetwork enriched for modifiers of neuronal development and morphology. To test the functional roles of these genes, we developed an experimental pipeline that allows pooled CRISPRi KD of 21 genes in human iPSC-derived neurons followed by optical analysis of genotypes, neuronal arborization, multiplexed pathway activity and morphological fingerprint readout. This approach recovered known genes involved in morphology and confirmed unexpected links from the network between several genetic modifiers of HD and morphology. Our approach overcomes challenges in pooled measurement of neuronal function and health and could be adapted for other phenotypes in HD and other neurological diseases.

neuroscience↗

bioRxiv2024

Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but not for live tissues due to its toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is minimally invasive to live cells, allowing for structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices, and the mouse brain in vivo. SeeDB-Live minimally affects the electrophysiological properties and sensory responses of neurons. We demonstrate its utility for widefield imaging of subcellular voltage dynamics, such as backpropagating action potentials, in acute brain slices. We also utilize SeeDB-Live for widefield voltage imaging of dozens of dendrites in vivo, demonstrating population dynamics. Thus, SeeDB-Live expands the scale and modalities of fluorescence imaging of live mammalian tissues.

neuroscience↗

bioRxiv2024

Harnessing RNA-based DNA repair pathways for targeted gene editing

Recent studies have revealed a role for RNA in the repair of DNA double-strand breaks. Here, we show that the asymmetric DNA overhangs generated by the small TevSaCas9 dual nuclease informs a simple and robust editing strategy in human cells whereby Pol{theta} and Rad52 are recruited to repair the double-strand break. The 2-nt, 3 DNA overhang generated by the I-TevI nuclease domain of TevSaCas9 hybridizes with the 3 end of a co-localized repair template guide RNA to specifically license repair. Substitutions that destabilize the repair duplex reduce editing efficiency. Targeted RNA-templated repair (rep-editing) harnesses cellular RNA-based DNA repair pathways to introduce precise nucleotide edits, deletions and insertions in human cells with high efficiency and fidelity independent of co-delivered repair functions. The small size of TevSaCas9 and RNA repair template offers delivery advantages over size-constrained or multi-component editing systems.

molecular biology↗

bioRxiv2020

A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila

Advances in CRISPR technology have immensely improved our ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. Here, we show that Cas13 works efficiently in Drosophila, both ex vivo and in vivo. We tested 44 different Cas13 variants to identify enzymes with the best overall performance and showed that Cas13 could target endogenous Drosophila transcripts in vivo with high efficiency and specificity. We also developed Cas13 applications to edit mRNAs and target mitochondrial transcripts. Our vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level.

molecular biology↗

bioRxiv2020

Gene expression profiling reveals subgenome dominance during Brassica napus seed development

We profiled the gene regulatory landscape of Brassica napus reproductive development using RNA sequencing. Comparative analysis of this nascent amphidiploid across the plant lifecycle revealed the contribution of each subgenome to plant reproduction. Global mRNA profiling revealed lower accumulation of Cn subgenome transcripts relative to the An subgenome. Subgenome-specific transcriptional networks identified distinct transcription factor families enriched in each of the An and Cn subgenome early in seed development. Global gene expression profiling of laser-microdissected seed subregions further reveal subgenome expression dynamics in the embryo, endosperm, and seed coat of early stage seeds. Transcription factors predicted to be regulators encoded by the An subgenome are expressed primarily in the seed coat whereas regulators encoded by the Cn subgenome were expressed primarily in the embryo. Data suggest subgenome bias are characteristic features of the B. napus seed throughout development, and that such bias might not be universal across the embryo, endosperm, and seed coat of the developing seed. Whole genome transcription factor networks identified BZIP11 as a transcriptional regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive-lethal phenotype. Taken together, transcriptional networks spanning both the An and Cn genomes of the B. napus seed can identify valuable targets for seed development research and that-omics level approaches to studying gene regulation in B. napus can benefit from both broad and high-resolution analyses. One Sentence SummaryGlobal RNA sequencing coupled with laser microdissection provides a critical resource to study subgenome bias in whole seeds and specific tissues of polyploid plants.

plant biology↗

bioRxiv2018

A Drosophila CRISPR/Cas9 toolkit for conditionally manipulating gene expression in the prothoracic gland as a test case for polytene tissues

Targeting gene function with spatial or temporal specificity is a key goal in molecular genetics. CRISPR-Cas9 has greatly facilitated this strategy, but some standard approaches are problematic. For instance, simple tissue-specific or global overexpression of Cas9 can cause significant lethality or developmental delays even in the absence of gRNAs. In particular, we found that Gal4-mediated expression of UAS-Cas9 in the Drosophila prothoracic gland (PG) was not a suitable strategy to disrupt gene expression, since Cas9 alone caused widespread lethality. The PG gland is widely used for studying endocrine gland function during animal development, but tools validating PG-specific RNAi phenotypes are lacking. Here, we present a collection of modular gateway-compatible CRISPR-Cas9 tools that allow precise modulation of target gene activity with temporal and spatial specificity. We also demonstrate that Cas9 fused to the progesterone ligand-binding domain can be used to activate gene expression via RU486. Using these approaches, we were able to avoid the lethality associated with simple GAL4-mediated overexpression of Cas9 in the PG. Given that the PG is a polytene tissue, we conclude that these tools work effectively in endoreplicating cells where Cas9 has to target multiple copies of the same locus. Our toolkit can be easily adapted for other tissues and can be used both for gain-and loss-of-function studies.

genetics↗