Biology subjects

Yin, J.-A.

Publications and source records attributed to Yin, J.-A..

13 recordsLinked to original sources

bioRxiv2026

PODXL regulates the neuronal entry of α-synuclein aggregates

Cell-to-cell transmission of -synuclein (-Syn) aggregates is a key event in Parkinson's disease (PD) pathogenesis, yet the neuronal surface factors that govern uptake of pathogenic -Syn fibrils remain undefined. We developed a flow cytometry-based CRISPR activation screen for modifiers of -Syn preformed fibril (PFF) uptake by human iPSC-derived cortical neurons. By interrogating 68 genes encoding cell-surface proteins prioritised by PD genetic associations, we identified podocalyxin-like protein 1 (PODXL) as a strong suppressor of -Syn fibril uptake. High-content imaging and PODXL cDNA overexpression confirmed reduced PFF uptake. Stable PODXL overexpression also reduced PFF-seeded pSynSer129 accumulation and uptake of structurally distinct recombinant and disease-derived -Syn fibrils. Conversely, shRNA-mediated PODXL knockdown increased -Syn PFF uptake, and analysis of PODXL domain-deletion and N-glycosylation mutants suggested that the ectodomain and N-glycosylation contribute to the PODXL-mediated reduction in uptake. Transcriptomic profiling further identified membrane metalloendopeptidase (MME) as a PODXL-responsive candidate mediator. MME activation and treatment with conditioned medium from MME-activated neurons reduced PFF uptake. Thiorphan, an MME inhibitor, partially attenuated the PODXL-associated reduction in uptake. Together, our findings identify PODXL as a neuronal brake on -Syn uptake and implicate its glycan-dependent surface properties together with MME-associated extracellular activity.

neuroscience↗

bioRxiv2026

Genome-wide arrayed CRISPR activation screen for prion protein modulators

The cellular prion protein (PrPC) is an essential substrate for prion propagation, and its abundance strongly influences susceptibility to prion disease. To systematically identify genetic regulators of PrPC abundance, we performed an arrayed genome-wide CRISPR activation (CRISPRa) screen targeting 19,839 human protein-coding genes in human glioblastoma cells. Using a quantitative time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, we measured PrPC levels across 22,442 individual genetic perturbations. This screen identified 531 genes whose activation significantly modulated PrPC abundance. A curated subset of 50 candidates was subjected to validation using independent TR-FRET assays and Western blotting, confirming 45 (90%) of tested hits. All raw and processed screening data, metadata, and analysis code are publicly available. This dataset provides a comprehensive resource for studying genetic regulation of PrPC homeostasis and enables reuse for investigations of membrane protein trafficking, proteostasis, and prion biology.

neuroscience↗

bioRxiv2025

Automation workflow for high-throughput arrayed plasmid DNA preparation and quantification.

High-throughput generation of arrayed plasmid DNA library using commercially available miniprep kits remains labor-intensive and costly. The yield and quality of plasmid preparations directly affect downstream applications, including arrayed viral library production and CRISPR library screening. Insufficient plasmid yield or DNA concentration often requires repeated preparations or additional DNA concentration steps to obtain adequate quantities. Similarly, higher variations in yield or quality across wells or plates can render an entire library unsuitable for subsequent experiments. To increase productivity and mitigate human intervention and errors, the present study established an automated workflow for high-throughput plasmid DNA preparation and quantification. The workflow was carried out by the Biomek i7 Hybrid automated workstation, synergizing a robotic liquid handler and multiple peripheral instruments to produce and measure plasmid DNA in a 96-well plate format. Bacterial competent cells were alkaline lysed and plasmid DNA was purified using magnetic beads, followed by quantification with the PicoGreen assay. The PicoGreen assay reported median and average yields of approximately 9.5 and 10 {micro}g per sample, respectively, which are equivalent to 7.6 and 8 {micro}g/mL of bacterial culture. Plasmid DNA concentrations measured by the PicoFluor fluorometer were consistently lower than those obtained using the NanoDrop UV spectrophotometer. The comparison demonstrated robust positive correlation between PicoGreen assay and NanoDrop measurements (R2 > 0.8). Among 480 plasmid DNA samples, average and median yields measured by the NanoDrop reached approximately 24 and 25 {micro}g per sample per well, corresponding to 19 and 20 {micro}g/mL of bacterial culture. Over 98% of samples exceeded the high-yield threshold of 10 {micro}g/mL of culture, with high plasmid quality validated through DNA gel electrophoresis. Collectively, this study demonstrated a robust, scalable, and cost-effective automation platform for high throughput arrayed plasmid library generation and quantification.

molecular biology↗

bioRxiv2025

Automation of high-throughput workflow for arrayed CRISPR activation library screening

CRISPR-mediated gene activation (CRISPRa) is among the most efficient and reliable strategies for mimicking sustained activation of endogenous promoters and their corresponding genes at physiological levels. By leveraging guide-RNA (gRNA) library design, CRISPRa screens can be applied on a whole-genome scale and are compatible with both arrayed and pooled formats, depending on assay requirements. Compared with conventional arrayed CRISPRa libraries that use single or dual gRNAs and often require multiple gRNA candidates per target, a recently developed CRISPRa library (termed T. gonfio) incorporates four tandem gRNAs per lentivector per target, thereby reducing library complexity and representing the smallest arrayed genome-wide CRISPRa library. To streamline genome-wide arrayed CRISPRa screening, this study developed a high-throughput automated workflow using the Biomek i7 Hybrid liquid-handling platform, integrated with multiple peripheral instruments. The workflow comprises three pipelines: lentiviral library transduction, cell library passaging, and assay processing. These pipelines together establish and maintain the transduced cell library for extended screening times. This enables assay processing at desired extended time points and improves the likelihood of identifying phenotypes that require longer time to develop, making the workflow suitable even for rapidly proliferating cell models. In a pilot arrayed screen using a T. gonfio mini-library targeting kinases and phosphatases, activation of the EPHA2 receptor promoter induced a growth reduction phenotype in the HEK293 cell model. This phenotype was recapitulated in a parallel pooled CRISPRa screen using the same mini-library and further validated in a co-culture assay.

cancer biology↗

bioRxiv2025

The Bone Morphogenetic Pathway Controls the Uptake of Infectious Prions

Prions propagate through cycles of intracellular replication, release, and uptake by host cells. Each of these steps is required for disease progression. Here, we used dCAS9-VP64 mediated transactivation to screen for determinants of prion uptake. We transduced PRNP-/- SH-SY5Y cells with a post-pooled human quadruple-guide-RNA library targeting all transcription start sites of the protein-coding genome, exposed them to synthetic infectious prions, and identified modifiers of prion internalization. Specificity was tested by counterscreens with other neurodegeneration-related aggregates and endocytic probes. Surprisingly, the Bone Morphogenetic Protein (BMP) signaling pathway emerged as a strong modulator. Prion uptake was increased by transcriptional activation of the BMPR1B, BMPR2 and ACVRL1 receptors, by the BMP effector SMAD1 and by exposure to BMP ligands. Conversely, prion uptake was reduced by activation of the BMP inhibitor SMAD6. Pharmacological inhibition of BMP signaling by the antagonistic ligand noggin and by the BMP kinase inhibitor dorsomorphin decreased prion internalization, reduced the number of prion-carrying cells, and suppressed the accumulation of proteinase-resistant prion protein in a panel of chronically infected human cell lines. Hence, cell-autonomous and non-autonomous BMP signaling components regulate prion uptake, and their manipulation provides a framework for interfering with the early steps of prion propagation.

neuroscience↗

bioRxiv2025

Automation of high-throughput arrayed lentivirus production and titration

Generation of arrayed genome-wide CRISPR libraries in a ready-to-transduce lentiviral format remains laborious, time-consuming, and costly. To address these limitations, the present study developed a fully automated lentivirus production and titration workflow using a Biomek i7 Hybrid automated workstation, integrated with multiple instruments and managed by SAMI EX software. The workflow produced and titrated viruses in 96 and 384-well plate formats, respectively. It employed reverse transfection and triplicate wells per lentivector to reduce variability and yielded an average of three viral particles in transduction unit (TU) per producing HEK293T cell. Titration was performed using U937-mCherry suspension cells, with the percentage of transduced cells converted from U937 (X%) to HEK293T (Y%) values via a linear regression equation (Y% = 4.3X% + 9.3%). The titer calculation was based on the initial seeding cell number, the converted percentage of HEK293T transduced cells, and virus input volume. The titration demonstrated strong reproducibility across LSRFortessa (BD) and Aurora (Cytek) flow cytometers (R2 = 0.9). Among 1,760 unconcentrated virus preparations, median and mean titers reached approximately 1.2 x 106 TU/mL, with over 97% of samples exceeding the high-titer threshold of 2x105 TU/mL, thus demonstrating a robust, scalable, and cost-effective automation platform for high throughput arrayed lentiviral library production and titration.

microbiology↗

bioRxiv2025

Automation of high-throughput arrayed mammalian cell line cultivation

Cell culture automation has traditionally been limited to basic tasks at low throughput, which are insufficient for passaging rapidly proliferating cell lines or for generating stable clonal lines. To address unmet needs, this study implemented a Biomek i7 Hybrid automated workstation, integrated with peripheral instruments and coordinated by SAMI EX software, to enable automated, high throughput mammalian cell culture workflows. The workflows support cell density monitoring, arrayed passaging, sample cherry-picking, plate reformatting, cell density normalization, and cryopreservation in 96-well plates. Integration with the CloneSelect imager allows rapid confluency monitoring and monoclonality assessment (<100 sec per plate). Cell passaging and density normalization require 32 minutes for one plate and 61 minutes for two plates. Workflow consistency was demonstrated across multiple cell lines and biological replicates, with wells showing comparable confluency within three standard deviations, lower coefficient of variation, and substantially narrower interquartile ranges after a single cell passage and density normalization. Four automation pipelines, including monoclonality screening, cell passaging and cherry-picking, density normalization, and cryopreservation, collectively enable clonal line establishment. Depending on scale, one to eight 384-well plates were processed in 69 to 355 minutes, yielding an average of 35 clonal lines per plate suitable for downstream genomic DNA sequence confirmation.

cell biology↗

bioRxiv2025

Large-scale bidirectional arrayed genetic screens identify OXR1 and EMC4 as modifiers of α-synuclein aggregation

In Parkinsons disease and other synucleinopathies, -synuclein (-Syn) misfolds and forms Ser129-phosphorylated aggregates (pSyn129). The factors controlling this process are largely unknown. Here, we used arrayed CRISPR-mediated gene activation and ablation to discover new pSyn129 modulators. Using quadruple-guide RNAs (qgRNAs) and Cas9, or an inactive Cas9 version fused to a synthetic transactivator, we ablated 2304 and activated 2428 human genes related to mitochondrial, trafficking and motility function in HEK293 cells. After exposure of cells to -Syn fibrils, pSyn129 signals were recorded by high-throughput fluorescence microscopy and aggregates were identified by image analysis. We found that pSyn129 was increased by activating the mitochondrial protein OXR1, which decreased ATP levels and altered the mitochondrial membrane potential. Instead, pSyn129 was reduced by ablation of the endoplasmic reticulum (ER)-associated protein EMC4, which enhanced ER-driven autophagic flux and lysosomal clearance. OXR1 activation preferentially modulated cellular reactions to fibrils derived from multiple system atrophy (MSA) patients, whereas EMC4 ablation broadly reduced pSyn129 across diverse -Syn polymorphs. These findings were confirmed in human iPSC-derived cortical and dopaminergic neurons, where OXR1 preferentially promoted somatic aggregation and EMC4 reduced both somatic and neuritic aggregates. These results uncover previously unrecognized roles for OXR1 and EMC4 in -Syn aggregation, thereby broadening our mechanistic understanding of synucleinopathies.

neuroscience↗

bioRxiv2024

TFAP2C and HNRNPK control mTOR cell metabolism and prion propagation

Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) is a limiting factor for prion propagation. However, little is known about the function of hnRNP K except that it is essential to cell survival. Here, we performed a synthetic-viability CRISPR ablation screen to identify epistatic interactors of HNRNPK. We found that deletion of Transcription Factor AP-2{gamma} (TFAP2C) suppressed the death of hnRNP K-depleted LN-229 and U-251 MG cells, whereas its overexpression hypersensitized cells to hnRNP K loss. HNRNPK ablation decreased cellular ATP, downregulated genes related to lipid and glucose metabolism, and enhanced autophagy. Co-occurrent deletion of TFAP2C reversed these effects, restoring transcriptional balance and alleviating energy deficiency. We linked HNRNPK and TFAP2C interaction to mTOR signaling, observing that HNRNPK ablation inhibited mTORC1 activity through downregulation of mTOR and Rptor, while TFAP2C overexpression enhanced mTORC1 downstream functions. In prion-infected cells, TFAP2C activation reduced prion levels and countered the increased prion propagation caused by HNRNPK suppression. Short-term pharmacological inhibition of mTOR also elevated prion levels and partially mimicked the effects of HNRNPK silencing. Our study identifies TFAP2C as a genetic interactor of HNRNPK, implicates their roles in mTOR metabolic regulation, and establishes a causative link between these activities and prion propagation.

cell biology↗

bioRxiv2024

Direct and indirect regulation of β- glucocerebrosidase by the transcription factors USF2 and ONECUT2

Mutations in the GBA gene, which encodes the lysosomal enzyme {beta}-glucocerebrosidase (GCase), are the most prevalent genetic susceptibility factor for Parkinsons disease (PD). However, only approximately 20% of carriers develop the disease, suggesting the presence of genetic modifiers influencing the risk of developing PD in the presence of GBA mutations. Here we screened 1,634 human transcription factors (TFs) for their effect on GCase activity in cell lysates of the human glioblastoma line LN-229, into which we introduced the pathogenic GBA L444P variant via adenine base editing. Using a novel arrayed CRISPR activation library, we uncovered 11 TFs as regulators of GCase activity. Among these, activation of MITF and TFEC increased lysosomal GCase activity in live cells, while activation of ONECUT2 and USF2 decreased it. Conversely, ablating USF2 increased GBA mRNA and led to enhanced levels of GCase protein and activity. While MITF, TFEC, and USF2 affected GBA transcription, ONECUT2 was found to control GCase trafficking by modulating the guanine exchange factors PLEKHG4 and PLEKHG4B. Hence, our study provides a systematic approach to identifying modulators of GCase activity, expands the transcriptional landscape of GBA regulation, and deepens our understanding of the mechanisms involved in influencing GCase activity.

molecular biology↗

bioRxiv2022

Robust and Versatile Arrayed Libraries for Human Genome-Wide CRISPR Activation, Deletion and Silencing

Arrayed CRISPR libraries extend the scope of gene-perturbation screens but require large numbers of efficacious sgRNA-expressing vectors. Using a newly invented liquid-phase plasmid cloning methodology, we constructed genome-wide arrayed libraries for human gene ablation (19,936 plasmids), activation, and epigenetic silencing (22,442 plasmids). At least 76% of each plasmid preparation encoded an intact array of 4 non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. We achieved perturbation efficacies of 75-99%, 76-92% and up to 10,000x in deletion, silencing and activation experiments, respectively. Upon conversion into massively parallel lentiviral vectors, an arrayed activation screen of 1,634 human transcription factors yielded 11 novel regulators of the cellular prion protein PrPC. Furthermore, a screen using a pooled version of the ablation library identified 5 novel modifiers of autophagy that went undetected with either of two 1sgRNA libraries. The CRISPR libraries described here represent a powerful resource for the targeted perturbation of human protein-coding genes.

genomics↗

bioRxiv2022

An Arrayed Genome-Wide Perturbation Screen Identifies the Ribonucleoprotein hnRNP K As Rate-Limiting for Prion Propagation

A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting modifiers of prion propagation. We exposed prion-infected cells in high-density microplates to 35364 ternary pools of 52746 siRNAs targeting 17582 genes representing the mouse protein-coding transcriptome. We identified 1191 modulators of prion propagation. While 1151 of these modified the expression of both the pathological prion protein, PrPSc, and its cellular counterpart PrPC, 40 genes affected selectively PrPSc. Of the latter, 20 genes augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC. Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Finally, the unexpected identification of a prioncontrolling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.

neuroscience↗

bioRxiv2021

The landscape of PrPC biosynthesis revealed by an arrayed genome-wide interference screen

The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64752 unique siRNAs targeting 21584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.

neuroscience↗