Biology subjects

Hsu, J. Y.

Publications and source records attributed to Hsu, J. Y..

11 recordsLinked to original sources

bioRxiv2024

Novel naphthyridones targeting Pannexin 1 for colitis management

Pannexin 1 (PANX1) forms cell-surface channels capable of releasing signaling metabolites for diverse patho-physiological processes. While inhibiting dysregulated PANX1 is proposed as a therapeutic strategy for many pathological conditions, including inflammatory bowel disease (IBD), low efficacy or poor specificity of classical PANX1 inhibitors introduces uncertainty for their applications in basic and translational research. Here, we performed hit-to-lead optimization and identified a naphthyridone, compound 12, as a new PANX1 inhibitor with an IC50 of 0.73 M that does not affect pannexin-homologous LRRC8/SWELL1 channels. Using structure-activity relationship analysis, mutagenesis, cell thermal shift assays, and molecular docking, we revealed that compound 12 directly engages PANX1 Trp74 residue. Using a dextran sodium sulfate mouse model of IBD, we found that compound 12 markedly reduced colitis severity, highlighting new PANX1 inhibitors as a proof-of-concept treatment for IBD. These data describe the mechanism of action for a new PANX1 inhibitor, identify the binding site for future drug design, and present a targeted strategy for treating IBD. TeaserA specific PANX1 inhibitor presents a proof-of-concept treatment for inflammatory bowel disease.

molecular biology↗

bioRxiv2024

CRISPR PERSIST-On enables heritable and fine-tunable human gene activation

Current technologies for upregulation of endogenous genes use targeted artificial transcriptional activators but stable gene activation requires persistent expression of these synthetic factors. Although general "hit-and-run" strategies exist for inducing long-term silencing of endogenous genes using targeted artificial transcriptional repressors, to our knowledge no equivalent approach for gene activation has been described to date. Here we show stable gene activation can be achieved by harnessing endogenous transcription factors (EndoTFs) that are normally expressed in human cells. Specifically, EndoTFs can be recruited to activate endogenous human genes of interest by using CRISPR-based gene editing to introduce EndoTF DNA binding motifs into a target gene promoter. This Precision Editing of Regulatory Sequences to Induce Stable Transcription-On (PERSIST-On) approach results in stable long-term gene activation, which we show is durable for at least five months. Using a high-throughput CRISPR prime editing pooled screening method, we also show that the magnitude of gene activation can be finely tuned either by using binding sites for different EndoTF or by introducing specific mutations within such sites. Our results delineate a generalizable framework for using PERSIST-On to induce heritable and fine-tunable gene activation in a hit-and-run fashion, thereby enabling a wide range of research and therapeutic applications that require long-term upregulation of a target gene.

molecular biology↗

bioRxiv2024

MOSAIC enables in situ saturation mutagenesis of genes and CRISPR prime editing guide RNA optimization in human cells

CRISPR prime editing offers unprecedented versatility and precision for the installation of genetic edits in situ. Here we describe the development and characterization of the Multiplexing Of Site-specific Alterations for In situ Characterization (MOSAIC) method, which leverages a non-viral PCR-based prime editing method to enable rapid installation of thousands of defined edits in pooled fashion. We show that MOSAIC can be applied to perform in situ saturation mutagenesis screens of: (1) the BCR-ABL1 fusion gene, successfully identifying known and potentially new imatinib drug-resistance variants; and (2) the IRF1 untranslated region (UTR), re-confirming non-coding regulatory elements involved in transcriptional initiation. Furthermore, we deployed MOSAIC to enable high-throughput, pooled screening of hundreds of systematically designed prime editing guide RNA (pegRNA) constructs for a large series of different genomic loci. This rapid screening of >18,000 pegRNA designs identified optimized pegRNAs for 89 different genomic target modifications and revealed the lack of simple predictive rules for pegRNA design, reinforcing the need for experimental optimization now greatly simplified and enabled by MOSAIC. We envision that MOSAIC will accelerate and facilitate the application of CRISPR prime editing for a wide range of high-throughput screens in human and other cell systems.

molecular biology↗

bioRxiv2023

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

cancer biology↗

bioRxiv2021

SIX1 is a master regulator of the Rhabdomyosarcoma undifferentiated state

Rhabdomyosarcoma (RMS) is a pediatric skeletal muscle sarcoma characterized by the expression of the myogenic-lineage transcription factors (TF) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their function. Here, we demonstrate that the developmental TF, SIX1, is highly expressed in RMS and is critical to maintain a muscle progenitor-like state. SIX1 loss induces terminal differentiation of RMS cells into myotube-like cells and dramatically impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over terminal muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status in RMS and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role for SIX1 in the repression of RMS differentiation via genome-wide alterations in MYOD1-mediated transcription. HighlightsO_LISIX1 prevents differentiation in RMS while it promotes differentiation during normal development C_LIO_LIFN-RMS are highly dependent on SIX1 for growth in both zebrafish and mouse xenograft models C_LIO_LILoss of SIX1 alters the transcriptional landscape of RMS cells, inducing a growth to differentiation switch C_LIO_LISIX1 knockdown in FN-RMS causes reduced super enhancer-based activity at stem-related genes and enhanced MYOD1 binding to differentiation loci, resulting in the activation of a myogenic differentiation program C_LIO_LIA gene signature derived from SIX1 loss strongly correlates with myogenic differentiation status and is predictive of advanced RMS. C_LI

cancer biology↗

bioRxiv2020

PrimeDesign software for rapid and simplified design of prime editing guide RNAs

Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we also constructed PrimeVar, the first comprehensive and searchable database for prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations to install or correct >68,500 pathogenic human genetic variants from the ClinVar database.

bioinformatics↗

bioRxiv2018

CRISPR-SURF: Discovering regulatory elements by deconvolution of CRISPR tiling screen data

Tiling screens using CRISPR-Cas technologies provide a powerful approach to map regulatory elements to phenotypes of interest, but computational methods that effectively model these experimental approaches for different CRISPR technologies are not readily available. Here we present CRISPR-SURF, a deconvolution framework to identify functional regulatory regions in the genome from data generated by CRISPR-Cas nuclease, CRISPR interference (CRISPRi), or CRISPR activation (CRISPRa) tiling screens. We validated CRISPR-SURF on previously published and new data, identifying both experimentally validated and new potential regulatory elements. With CRISPR tiling screens now being increasingly used to elucidate the regulatory architecture of the non-coding genome, CRISPRSURF provides a generalizable and accessible solution for the discovery of regulatory elements.

bioinformatics↗

bioRxiv2018

STREAM: Single-cell Trajectories Reconstruction, Exploration And Mapping of omics data

Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data.

genomics↗

bioRxiv2017

BrainImageR: Spatiotemporal gene set analysis referencing the human brain

RATIONALENeurological molecular analyses such as transcriptomics, epigenetics, and genome-wide association studies must be assessed in the context of the human brain in order to generate biologically meaningful inferences. It is often difficult to access primary human brain tissue; therefore, approximations are made using in vitro modeling or by identifying disease-associated genes from DNA extracted from blood. Gene sets from these studies are then compared to the post-mortem human brain to provide an assessment of the brain region and the developmental time point that a gene set is most closely associated with. However, most analyses of postmortem datasets are achieved by building new computational tools each time in-house, which can cause discrepancies from study to study, indicating that the field is in need of a user-friendly suite of tools to examine spatiotemporal expression with respect to the postmortem brain. Such a tool will be of use to the molecular interrogation of neurological and psychiatric disorders, with direct advantages for the disease-modeling and human genetics communities.\n\nRESULTSWe have developed brainImageR, an R package that calculates both the spatial and temporal association of a dataset with post-mortem gene expression data from the Allen Brain Atlas. BrainImageR performs a robust analysis of gene set enrichment to identify enriched anatomical regions, provides a global high-resolution visualization of these enrichments across the human brain, and predicts when in developmental time the sample is most closely matched to, a task that has become increasingly important in the field of in vitro neuronal modeling. These functionalities of brainImageR enable a quick and efficient characterization of a given dataset across normal human brain development.\n\nAVAILABILITY AND IMPLEMENTATIONBrainImageR is released under the Creative Commons CC BY-SA 4.0 license and the source code can be downloaded through github at https://github.com/saralinker/brainImageR.\n\nCONTACTgage@salk.edu

bioinformatics↗

bioRxiv2017

"Unexpected mutations after CRISPR-Cas9 editing in vivo" are most likely pre-existing sequence variants and not nuclease-induced mutations

Schaefer et al. recently advanced the provocative conclusion that CRISPR-Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site.1 Using high-coverage whole genome sequencing (WGS), these authors reported finding SNPs and indels in two CRISPR-Cas9-treated mice that were not present in a single untreated control mouse. On the basis of this association, Schaefer et al. concluded that these sequence variants were caused by CRISPR-Cas9. This new proposed CRISPR-Cas9 off-target activity runs contrary to previously published work2-8 and, if the authors are correct, could have profound implications for research and therapeutic applications. Here, we demonstrate that the simplest interpretation of Schaefer et al.s data is that the two CRISPR-Cas9-treated mice are actually more closely related genetically to each other than to the control mouse. This strongly suggests that the so-called \"unexpected mutations\" simply represent SNPs and indels shared in common by these mice prior to nuclease treatment. In addition, given the genomic and sequence distribution profiles of these variants, we show that it is challenging to explain how CRISPR-Cas9 might be expected to induce such changes. Finally, we argue that the lack of appropriate controls in Schaefer et al.s experimental design precludes assignment of causality to CRISPR-Cas9. Given these substantial issues, we urge Schaefer et al. to revise or re-state the original conclusions of their published work so as to avoid leaving misleading and unsupported statements to persist in the literature.

molecular biology↗

bioRxiv2017

Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors

Targeted and inducible regulation of mammalian gene expression is a broadly important research capability that may also enable development of novel therapeutics for treating human diseases. Here we demonstrate that a catalytically inactive RNA-guided CRISPR-Cpf1 nuclease fused to transcriptional activation domains can up-regulate endogenous human gene expression. We engineered drug-inducible Cpf1-based activators and show how this system can be used to tune the regulation of endogenous gene transcription in human cells. Leveraging the simpler multiplex capability of the Cpf1 platform, we show that we can induce both synergistic and combinatorial gene expression in human cells. Our work should enable the creation of other Cpf1-based gene regulatory fusion proteins and the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.

molecular biology↗