Biology subjects

Yu, T.

Publications and source records attributed to Yu, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Multiple Pol determinants constrain koala retrovirus A replication

Koala retrovirus subtype A (KoRV-A) is a gammaretrovirus that occurs in both endogenous and horizontally transmissible forms in wild koala populations. KoRV infection is associated with neoplasia and increased susceptibility to opportunistic infections. Its ongoing incorporation into the koala germline provides a rare contemporary example of retroviral endogenization in a mammalian host. We generated a consensus KoRV-A clone using viral sequences from an infected wild koala and compared it with a published clone representing a viral isolate from a captive koala. Both replicated far less efficiently than Moloney murine leukemia virus (Mo-MLV), the best-characterized gammaretrovirus, during spreading infection in human cell lines, prompting us to investigate the viral features underlying this difference. Following transfection, KoRV-A and Mo-MLV produced comparable levels of viral RNA and intracellular Gag and Pol proteins, yet KoRV-A yielded approximately 1,000-fold less particle-associated reverse transcriptase (RT) activity and markedly lower amounts of Gag- and Pol-derived proteins in released virions. Reduced accumulation of extracellular viral proteins persisted with protease-inactive precursors, indicating that impaired proteolytic maturation alone could not explain this phenotype. KoRV-A/Mo-MLV chimeras showed that particle-associated RT activity depended on both RT sequence and compatibility between protease (PR) and the cleavage sites flanking RT; replacement of the entire KoRV-A PR-RT region with Mo-MLV sequence restored RT activity to near Mo-MLV levels. Transmission electron microscopy (TEM) and spatial analysis revealed that KoRV-A virions accumulated in substantially larger and denser extracellular clusters than Mo-MLV virions. RT and PR-RT chimeras exhibited increased particle abundance and apparent clustering, although most spatial differences were attenuated after accounting for particle density. Whether extracellular particle aggregation contributes directly to inefficient replication remains unclear. Together, these findings identify coordinated interactions among PR, RT, and their cleavage-site context as determinants of KoRV-A enzymatic activity and reveal an unusual extracellular particle organization associated with a gammaretrovirus.

microbiology↗

bioRxiv2026

Active maintenance of accessible chromatin at poised meiotic promoters by the NFYA-NURF axis is required for meiotic entry in mice

Developmental genes often acquire accessible chromatin prior to their activation, yet the mechanisms regulating this permissive state remain poorly understood. During spermatogenesis, many meiotic gene promoters display this poised state in spermatogonia, characterized by accessible promoters and paused RNA Pol II occupancy before their activation during meiosis. Here, we identify the NURF chromatin remodeling complex, comprised of SMARCA5 and its accessory subunit BPTF, as a key regulator of this process. We demonstrate that the transcription factor NFYA interacts with SMARCA5 and is required for its occupancy at poised meiotic gene promoters. Approximately one-third of poised meiotic gene promoters is co-occupied by NFYA, SMARCA5, and BPTF. Germline deletion of Smarca5 disrupts chromatin accessibility at poised meiotic promoters, impairs transcriptional program required for mitosis-to-meiosis transition, resulting in developmental arrest at meiotic entry. These findings identify NFYA-NURF as a key regulatory axis that regulates accessible chromatin at developmentally poised meiotic genes and primes them for activation later during meiosis.

developmental biology↗

bioRxiv2026

A single-nucleus multi-omic atlas of gene regulation across 21 adult human tissues

Diverse human cell types establish specialized functions through lineage- and context-specific regulatory programs. Interpreting non-coding genetic risk requires integrated multi-omic reference maps that directly connect regulatory DNA to cellular expression across human tissues. Here we present a single-nucleus multi-omic atlas comprising 459,856 transcriptomic and chromatin accessibility profiles from 21 adult human tissues and four donors, including paired measurements from 160,688 nuclei. The atlas resolves nine cell lineages, 61 broad cell types and 313 subclusters, and identifies 1,085,062 candidate cis-regulatory elements (cCREs), including 161,270 novel elements absent from ENCODE. Regulatory activity was dominated by cell identity but refined by tissue context. Joint profiling enabled 871,177 cCRE-gene associations and revealed lineage-specific regulatory architectures. Cross-tissue accessibility further identified lineage-restricted and constitutively inaccessible chromatin domains, the latter showing preferential hypomethylation across human cancers. Furthermore, we leverage this dataset to train sequence-to-function models to predict chromatin-accessibility effects for 548,656 fine-mapped variants, identifying 18,133 high-effect variants, including 1,120 broadly active variants. Models trained for eight endothelial subtypes further resolve predicted variant effects across vascular beds. Together, this atlas provides a comprehensive cellular and computational framework for interpreting regulatory sequence, context-dependent gene control, and complex trait genetics across the human body.

genomics↗

bioRxiv2026

Evolutionarily labile pachytene piRNAs target an altered set of mRNAs in male hybrids of house mouse subspecies

During male meiosis-I of placental mammals, [~]30-nucleotide pachytene PIWI-interacting RNAs (piRNAs) are expressed to regulate genes required for sperm function. Pachytene piRNA genes evolve rapidly. Whether rapid evolutionary turnover of pachytene piRNAs is under positive selective pressure remains enigmatic. Here, we investigate the evolutionary rate of pachytene piRNA genes over a short evolutionary timescale using geographically isolated mouse subspecies. We demarcate the genes producing postnatal piRNAs in PWK/PhJ and CAST/EiJ. Comparative genomics reveals 16 subspecies-specific pachytene piRNA loci underscoring how labile pachytene piRNA genes are even during short evolutionary timescale. We report a highly abundant CAST/EiJ-specific pi17-CAST locus defying the notion that young pachytene piRNA genes do not produce abundant piRNAs. In fact, male hybrids from the reciprocal crossing C57BL/6J and CAST/EiJ produce pi17-CAST piRNAs almost exclusively from the CAST/EiJ allele suggesting that species-specific nucleotide variants are sufficient to turn a locus into piRNA source. Intriguingly, hybrid males with reduced fertility features retain distinct piRNA-mRNA pairs compared to parents. Our work reveals that rapidly evolving pachytene piRNAs can gain or lose targets in the hybrid males of closely related mammalian species.

evolutionary biology↗

bioRxiv2026

A novel SXXLF motif in the FXR N-terminal domain mediates coregulator and interdomain interactions

The nuclear receptor superfamily is comprised of ligand-regulated transcription factors that contain an intrinsically disordered domain at the amino-terminal end, known as the N-terminal domain (NTD). While this poorly conserved domain is known to possess ligand-independent activation function (AF-1), few NTD functions are conserved between nuclear receptors (NRs). Identified roles in other receptors include androgen receptor (AR), estrogen receptor (ER) and mineralocorticoid receptor (MR). Here, we aim to define the function of the NTD of the farnesoid X receptor (FXR), a crucial regulator of lipid and bile acid metabolism. We show that the NTD engages in interdomain contact with other FXR domains. We also observe that the NTD interacts directly with coregulator proteins. Using mutagenesis, mammalian two-hybrid assays and molecular dynamics simulations, we identify and validate a novel SXXLF motif in the NTD which mediates interactions with both coregulators and the ligand binding domain. Mutation of the motif induces large changes in conformational and allosteric coupling in FXR. Our study identifies a new nuclear receptor-interacting motif that modulates the transcriptional activity of FXR. Graphical AbstractFXR-NTD regulates transcriptional activity through interdomain communication with the LBD and is also involved in co-activator recruitment. The SENLF motif is the first defined functional element within the FXR-NTD and mediates both NTD-LBD interaction and selective co-activator engagements to drive NTD-mediated transcriptional activity. O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/724725v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5a37aorg.highwire.dtl.DTLVardef@2fa9e1org.highwire.dtl.DTLVardef@13a19daorg.highwire.dtl.DTLVardef@1775ed2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Neuromodulation of Foxp2+ hypothalamic neurons induces therapeutic hypothermia

The inability to selectively trigger therapeutic hypothermia independent of environmental cooling has hindered causal analysis of its broader physiological benefits. Here, we employed P57, a natural product that pharmacologically induces therapeutic hypothermia circumventing external cold stress, to identify the neuronal substrates underlying hypothermia-induced antitumor effects. Integrating functional ultrasound imaging, activity-dependent neuronal labeling, and single-nucleus RNA sequencing, we identify a previously unrecognized population of Foxp2 neurons in the medial preoptic area (MPA) that are selectively activated by P57. Inhibition of MPA Foxp2+ neurons abolishes P57-induced hypothermia, whereas the paraventricular hypothalamus (PVH) serves as a critical downstream node. Importantly, targeted activation of MPA Foxp2 neurons is sufficient to induce sustained hypothermia, suppress systemic metabolism, and inhibit tumor growth, providing causal evidence that neuronally driven reductions in core body temperature can exert antitumor effects. Together, these findings establish MPA Foxp2 neurons as a controllable node for therapeutic hypothermia induction and demonstrate that neuromodulation of defined neuronal populations can achieve therapeutic benefit by directly controlling physiological states.

neuroscience↗

bioRxiv2026

Cohesin extrudes chromatin loop unidirectionally through two modes of mechanisms in human cells

Cohesin is crucial for establishing 3D genome organization and functions. However, the in vivo mechanism of cohesin-mediated extrusion remains unsettled. Here, we investigated extrusion directionality using integrated genome mapping coupled with perturbation in human cells. By defining cohesin loading at NIPBL binding loci and anchoring at CTCF sites, we systematically characterized the aggregated and single-molecule trajectories of extrusion "stripes" and contact "dots" from and between individual cohesin loading and anchoring points. Our analyses delineated two dynamic modes of in vivo mechanism in which NIPBL/cohesin initiates extrusion at its loading sites in a one-sided manner; after encountering CTCF, CTCF/cohesin switches its extrusion direction backward and robustly extrude DNA unidirectionally until completing the convergent CTCF loop. Surprisingly, depleting CTCF resulted substantial increase of NIPBL/cohesin-mediated extrusion activity, which is further validated by in silico perturbation. These findings reveal novel mechanistic insights into how cohesin and CTCF orchestrate cohesin-mediated loop extrusion in vivo.

genomics↗

bioRxiv2026

Metabolic signals regulate resuscitation speed of antibiotic persister bacteria during infection

All living organisms adjust their metabolism in response to environmental changes. Under unfavorable conditions, organisms enter a state of dormancy by halting metabolism, enabling survival. Dormant bacteria become highly tolerant to antibiotics-a phenomenon called persistence. Here, we demonstrate that selective metabolic reprogramming controls the resuscitation speed of persister after antibiotic exposure. Using multi-omics and in silico modeling, we found that dormant bacteria reprogram metabolic pathways to modulate persister awakening. Accumulation of L-serine and reduction of arginine drive rapid resuscitation. L-serine promotes cysteine biosynthesis and motility while reducing energy metabolism to facilitate rapid resuscitation. In contrast, arginine slows regrowth from dormancy by enhancing ethanol-aldehyde and energy metabolism. L-serine and arginine can, respectively, promote or inhibit the regrowth of antibiotic persister cells in macrophages and mouse models, and regulate the awakening speed of Salmonella, E. coli, and methicillin-resistant Staphylococcus aureus (MRSA). These findings suggest new strategies to target chronic bacterial infections. TeaserL-serine speeds and arginine slows the awakening of antibiotic persisters, revealing targets for chronic infection.

microbiology↗

bioRxiv2026

Coenzyme A is a redox sensing cofactor for malic enzyme 2 regulating oxidative stress and mitochondrial metabolism

Coenzyme A (CoA) is an essential cofactor required for numerous metabolic reactions, yet its ability to bind and regulate proteins remains poorly defined. Using a proteomic approach, we identified malic enzyme 2 (ME2) as a CoA-binding protein. ME2 uses NAD(P)+ to convert malate to pyruvate, generating NAD(P)H to support energy production and redox homeostasis. ME2 binds CoA at an allosteric site previously thought to bind NAD(P)+. Reduced CoA has minimal effect on ME2 activity, but the oxidized form, CoA disulfide, strongly activates ME2 by promoting ME2 tetramerization and a catalytically efficient closed conformation. Under oxidative stress, ME2 facilitates CoA disulfide formation, enhancing NADPH production and cellular defense against reactive oxygen species (ROS). Mice with ME2 mutation that cannot bind CoA show impaired muscle performance, elevated ROS, and mitochondrial dysfunction. These findings establish CoA as a redox-sensing cofactor, allowing cells to respond to ROS and promote mitochondrial metabolism, and expanding the function of this essential cofactor.

biochemistry↗

bioRxiv2026

Multiscale Mechanisms of Human Memory Modulation by Deep Brain Stimulation

The highly variable effects of deep brain stimulation (DBS) on cognition highlight a fundamental gap in our understanding of how external perturbations interface with the brains ongoing, task-engaged state. The current study addressed this challenge by combining intracranial EEG recordings with hippocampal DBS stimulation in human participants during spatial sequence memory task. We found 50 Hz stimulation targeting hippocampal white matter enhanced memory performance, whereas 5 Hz stimulation targeting hippocampal gray matter impaired memory. These opposing behavioral outcomes were both governed by two dissociated mechanisms: a region-specific modulation of theta rhythms in an engagement-dependent fashion, and a global modulation of the fidelity of memory representations across the cortex. Our results suggest targeted stimulation can concurrently fine-tune specialized circuits and govern the brains global state, which not only offers a novel roadmap for understanding how external inputs shape human cognition, but also helps to design precise, state-dependent neuromodulation therapies.

neuroscience↗

bioRxiv2026

Structure-guided discovery and engineering of miniature CRISPR-Cas12m for epigenome editing

CRISPR-based epigenome editing represents a programmable strategy to precisely modulate gene expression, holding immense promise for therapeutic applications. However, the large size of the dCas proteins substantially impedes the delivery via adeno-associated virus (AAV) vectors. Here, through iterative bioinformatics analysis, structure-guided predictions, and functional assays, we identified and characterized PmCas12m, a novel miniature subtype V-M CRISPR-Cas12m. PmCas12m exhibited flexible 5-YTN-3 PAM-dependent recognition and robust double-stranded DNA binding properties, while lacking DNA cleavage activity, thus positioning it as an ideal tool for epigenome editing. Cryogenic electron microscopy (cryo-EM) structures of PmCas12m unveiled its unique molecular mechanism of DNA binding facilitating interference. Guided by these structural insights, we employed deep mutational scanning (DMS) and protein engineering to develop xCas12m, a hypercompact variant with highly potent and specific epigenome editing capabilities in human cells. We further constructed the xCas12m-CRISPRoff platform in a single AAV vector, which achieved durable epigenetic silencing and effective inhibition of hepatitis B virus (HBV) infection in a mouse model. Collectively, these findings establish xCas12m as a versatile epigenome editing platform with transformative potential for treating diseases, paving the way for clinical translation of epigenetic therapies.

bioengineering↗

bioRxiv2026

Postnatal gene restoration in succinic semialdehyde dehydrogenase deficiency (SSADHD) reveals phenotype reversibility

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive metabolic disorder due to loss-of-function ALDH5A1 mutations impairing the catabolism of {gamma}-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain. In SSADHD, pathologic accumulation of GABA and its metabolic by-product {gamma}-hydroxybutyrate (GHB) corresponds to a clinical syndrome dominated by developmental delay and epilepsy in half of patients with risk of sudden death in adolescence and adulthood. Brain-wide ALDH5A1 gene replacement for SSADHD is unavailable, and whether such treatment will reverse the SSADHD phenotype is unknown. We developed an inducible mouse SSADHD model, Aldh5a1lox-STOP, enabling Cre-dependent Aldh5a1 restoration to evaluate gene therapy feasibility. In the absence of SSADH, Aldh5a1lox-STOP mice exhibit hyperactivity and excessive serum GHB levels, culminating in death by [~]postnatal day 22, recapitulating the severe SSADHD condition. Systemic delivery of a blood-brain barrier (BBB)-penetrating adeno-associated virus (AAV) carrying a Cre gene to Aldh5a1lox-STOP mice leads to brain-wide SSADH restoration, serum GHB level reduction, normalization of hyperactivity, and substantial increase in survival. As a step toward clinical translation, we further assessed an AAV encompassing a functional native promoter (FLnP) of ALDH5A1 tethered to its human coding sequence, namely AAV-FLnP-hALDH5A1. Aldh5a1lox-STOP mice were effectively rescued when treated with AAV-FLnP-hALDH5A1 packaged in the blood-brain barrier (BBB)-penetrating capsid PHP.eB. These findings provide preclinical proof that SSADH gene replacement therapy is feasible and potentially effective.

neuroscience↗

bioRxiv2026

EnzySeek: Efficient Exploration of Enzyme Reaction Pathways Using AI Agents

With the rapid development of Large Language Models (LLMs) and Agent technologies, AI can assist in solving a variety of real-world problems across multiple domains, such as autonomous driving, drug discovery, and materials design. In this work, we present EnzySeek, an enzyme catalysis AI agent designed to assist researchers in enzyme catalysis simulations. First, we constructed a domain-specific knowledge base by curating thousands of papers related to enzyme catalysis. Second, we customized Model Context Protocol (MCP) interfaces for each step of the enzyme catalysis simulation workflow, enabling these functions to be invoked by LLMs. Finally, we configured an agent capable of simultaneously referencing past empirical studies on enzyme catalysis, autonomously executing tool calls, and analyzing as well as presenting the results. EnzySeeks capabilities cover multiple aspects, including protein structure prediction, molecular docking, system preparation and parameterization, molecular dynamics (MD) simulations, and QM/MM calculations. The conclusions drawn by EnzySeek are primarily based on the results of QM/MM calculations. We employed the semi-empirical quantum mechanical method GFN2-xTB to calculate the QM region of the system. Benchmark results indicate that the GFN2-xTB method can achieve high efficiency while maintaining accuracy. The EnzySeek agent is designed to continuously learn from newly published literature and past computational tasks. During its operation, every AI decision is manually verified and scored by human experts. This human-in-the-loop validation provides the AI with sufficient case-based support, ultimately contributing to the full automation of enzyme catalysis computations. All data generated during the simulations are compiled into a dataset, which is used to establish evaluation criteria specific to enzyme catalysis computational results.

biochemistry↗

bioRxiv2026

Viral haplotype reconstruction from long reads with virCHap

Resolving genomes at the haplotype level for viral populations is crucial for understanding the prevalence of viral diseases and for the development of effective therapeutic treatments. However, viral haplotype reconstruction still presents challenges, such as an unknown number of strains, high inter-strain similarity, repetitive regions, and difficulties in abundance estimation. Here, we developed virCHap, a new reference-based haplotype phasing algorithm for viruses, which applies graph partitioning followed by iteratively quantifiable cluster merging on long-read sequencing data. Benchmarking on simulated and real datasets demonstrates that virCHap outperforms current tools in terms of recall, accurate abundance estimates and read clustering accuracy. On the simulated large-genome VZV experiment, virCHap had a 96.5% recall, 14% higher than the second-best method, and had the most accurate abundance estimates. On a real 5-strain PVY dataset, virCHap had a precision exceeding 92.9%, a recall of over 97%, and a read clustering accuracy of 82%, outperforming the second-best method by 32%. On a real 6-strain SARS-CoV-2 dataset, virCHap achieved >96.9% accuracy, and the most accurate abundance estimates within the spike gene.

bioinformatics↗

bioRxiv2026

Lets talk about sex: Male and female mice show similar fear memory retention despite hippocampal activity differences during encoding and consolidation

Accurate and efficient memory processing is essential for survival. A body of ongoing work in both human subjects and animal models suggests that memory processing may differ substantially between males and females. In mice, contextual fear memory (CFM) encoding, consolidation, and recall have been well studied, and the mouse hippocampus and amygdala have been implicated in these processes. The present study addresses whether the activation of these brain regions differs between male and female mice at each stage of CFM processing. We find that male and female C57BL6 mice show no differences in sleep behavior following single-trial contextual fear conditioning (CFC), which is essential for CFM consolidation. We also find no significant differences in CFM recall behavior between male and female mice. However, females - but not males - show significantly increased expression of cFos in dorsal hippocampal CA1 and CA2 neurons during CFM encoding. On the other hand, males - but not females - show increased cFos expression among dentate gyrus (DG) granule cells, and decreased cFos expression within dorsal CA1 and CA3 during CFM consolidation. Males also show significant decreases in dorsal CA1 cFos expression during memory recall. These findings highlight the idea that the neurobiological underpinnings of memory processing may differ between males and females, even when recall performance is identical. HighlightsHistorically, research on the neurobiological basis of memory processing has been carried out mainly in male subjects. Thus, our understanding of these mechanisms is biased towards male brain neurophysiology. This is problematic, as numerous studies have reported sex-based performance differences for episodic memory tasks, in which male subjects have variously performed better, worse, or the same as females. Here, we find that male and female mice perform similarly on a well-studied experimental memory task but nonetheless have differences in the relative activity of different brain structures during sequential stages of memory processing. This emphasizes the importance of including both males and females in memory studies, due to potential sex differences in the neurobiological substrates of memory.

neuroscience↗

bioRxiv2025

A novel dephosphorylation peptide inhibits 17β-HSD1 enzyme activity in ovarian granulosa cells and breast cancer cells

Estradiol (E2), a pivotal mammalian reproductive hormone, is closely associated with estrogen-dependent diseases. 17{beta}-hydroxysteroid dehydrogenase type 1 (17{beta}-HSD1), a critical E2 synthesis enzyme, has been understudied for its post-translational regulation-despite its potential as a therapeutic target, no clinically approved inhibitors are currently available. In our investigation of porcine follicular atresia mechanisms, we identified five differentially phosphorylated residues in 17{beta}-HSD1. Using in vitro site-directed mutagenesis and transgenic mouse models carrying point mutations, we established that Ser30 and Ser274 are critical phosphorylation sites that robustly modulate 17{beta}-HSD1 enzymatic activity. We further demonstrated that activin A and insulin-like growth factor 1 (IGF-1) enhance 17{beta}-HSD1 activity by promoting its phosphorylation at these two sites. To target these regulatory residues, we designed and synthesized cell-penetrating peptides (CPPs) that specifically suppress 17{beta}-HSD1 phosphorylation. Functional assays conclusively demonstrated that these CPPs significantly suppressed 17{beta}-HSD1 activity in vitro. Notably, they also inhibited the proliferation, migration and invasion of MCF-7 cells, as well as tumor growth in a mouse model of breast cancer. Our study provides novel mechanistic insights into the regulation of 17{beta}-HSD1 and E2 synthesis, addressing a critical gap in steroid hormone biology. The identification of Ser30/Ser274 as functional phosphorylation sites and the development of CPP-based inhibitors offer both theoretical advances and translational potential, opening new avenues for the treatment of estrogen-dependent breast cancer.

cell biology↗

bioRxiv2025

tBid mediated genetic ablation of connective tissue cells reveal their key regulatory function during limb regeneration in axolotls

Limb regeneration in adulthood is a fascinating phenomenon unique to salamanders, requiring precise coordination and interaction of various cell types. Connective tissue (CT) constitutes a major component of the limb and serves as the primary reservoir of positional information during regeneration. However, the direct evidence and the extent of CT in positional contribution remain limited. Here we develop an inducible Cre-LoxP-mediated tBid cell ablation system in axolotls and demonstrate efficient elimination of targeted muscle tissue and muscle stem cells through transient electroporation or genetic approach. Ablation of CT cells at early stages of regeneration results in delayed regeneration and loss of proximal limb segments (e.g., upper and lower limb), with minimal impact on hand differentiation. CT ablation during development yields similar defects as observed during regeneration, indicating an essential role for CT cells in determining the positional identity of developing and regenerating limb structures. Further scRNA-seq reveal a progressive proximal-to-distal transition among CT cells and identify distinct CT subtypes that contribute to proximal and distal segments. CT ablation results in reduction of the differentiated CT1--a subpopulation that maintains proximal identity throughout regeneration, and a delay of distal transformation in the proliferative CT1--a major CT subtypes showing progenitor property. These cellular shifts likely lead to the observed morphological deficits in regenerating limbs after CT ablation. Moreover, differentiated CT1 cells enhanced their interactions with surrounding cell types after CT ablation, to modulate adaptive proliferation in other populations, e.g., muscle stem cells. Our work establishes a tBid-based ablation strategy for functional studies of CT and other cell types in axolotls, and provides direct evidence demonstrating pivotal roles of CT cells in guiding positional memory and segmental patterning during organ regeneration.

molecular biology↗

bioRxiv2025

LDLR variant classification through activity-normalized prime editing screening

BackgroundInherited variants in the LDL receptor (LDLR) gene are the most common cause of familial hypercholesterolemia (FH), significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt intervention with lipid-lowering therapies; however, the majority of LDLR variants observed in the population have uncertain or absent clinical classifications, limiting the potential to improve clinical management. MethodsWe developed an innovative, activity-normalized prime editing screening pipeline to measure the impact of 5,184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Through pairing a genotypic outcome reporter with every prime editing guide RNA (pegRNA), we adjust phenotypic measurements to account for variable editing efficiency, extending activity normalization to prime editing for the first time at this scale. Further, we use a statistical estimation approach that leverages measurements for all missense variants at a given position to denoise the resulting scores. ResultsWe show that prime editing-mediated reporter editing correlates with endogenous variant installation frequency, allowing activity normalization to improve imputation of LDLR variant effect. Our optimized prime editing assay identifies a broad, continuous spectrum of variant functional effects. We achieve robust separation of pathogenic vs. benign ClinVar variants and concordance between experimentally derived functional scores and LDL-C levels measured in UK Biobank participants. Further, when calibrating the strength of evidence provided by this functional screening data to align with the ACMG/AMP variant interpretation guidelines, and integrating additional sources of evidence, a majority of currently unclassified rare LDLR variants meet evidence thresholds for reclassification. We use the broad coverage of this screen to gain insight into how apolipoproteins bind to LDLR. In particular, we identify and characterize rare LDLR variants that enhance LDL-C uptake through increased interaction with apolipoprotein B. Finally, we compare prime editing-based functional scores with those derived from recent base editing and cDNA-based LDLR variant screens, showing that these approaches all show robust correlation with clinically observed LDL-C levels and computational scores, while prime editing identifies candidate splice-altering coding variants that are not modeled by cDNA screening. ConclusionsAltogether, our approach demonstrates the power of prime editing to significantly improve understanding of how variants in LDLR impact function and contribute to FH.

genetics↗