Biology subjects

Corn, J. E.

Publications and source records attributed to Corn, J. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Predicting cellular functions of human phosphosites

Protein phosphorylation is a regulatory switch that controls essentially all cellular processes, including metabolism, cell cycle progression, and DNA repair. Furthermore, by modulating specific regions and activities of a protein, phosphorylation sites can independently coordinate the proteins involvement in distinct cellular processes. Despite the importance of this regulatory mechanism, the majority of human phosphosites have not been functionally characterized. Here we have developed biologically informed predictors of phosphosite cellular function by integrating protein functional annotation, recognition by upstream kinases, and regulation across phosphoproteomics perturbations. To enable this we harmonized public phosphoproteomics data into an atlas of nearly 2,000 perturbations and more than 16 million quantifications, providing a powerful resource for studying phosphosite regulation. We applied our predictors to functionally characterise thousands of understudied phosphosites, revealing many phosphosite functions that could not be inferred from protein functional annotation, and functionally divergent phosphosites within the same protein. Using cancer multi-omics data we characterized connections between these phosphosites and other molecular layers. Finally, we illuminated novel signaling axes within the DNA repair pathway, and demonstrated that phosphosites on PPM1G and KHSRP modulate cell survival under DNA damage. Our work represents a powerful resource for dissecting the cellular consequences of protein phosphorylation.

genomics↗

bioRxiv2025

DCLRE1A orchestrates CAG repeat contraction following Cas12a-induced DNA breaks

Despite their well-defined genetic background, repeat expansion diseases (REDs) still represent an unmet medical need, with no causative therapy offered to patients. The strategy of repeat shortening using genome editing tools is very attractive because a single intervention can result in permanent repair of the disease-causing mutation. However, a limited understanding of DNA repair mechanisms in repetitive sequences complicates the prediction and control of the editing effects. Using a CRISPR interference (CRISPRi) screen, we identified pathways and factors responsible for the repair of staggered cuts generated by Cas12a within CAG repeat tracts. This analysis revealed a central role for interstrand crosslink (ICL) repair factors in mediating CAG repeat contraction, with DCLRE1A emerging as a key effector. We demonstrated that DCLRE1A recognizes and binds structures generated by Cas12a. Moreover, DCLRE1A interacts with SLX4, promoting the generation of pure contractions, and with POLI, leading to the formation of inverted repeats at the break site during template switching mechanisms. We then used this knowledge to increase the contribution of pure contractions to the pool of editing outcomes using fusions of Cas12a with DNA repair proteins. Our study indicates that Cas12a can be used as an effective tool for generating repeat contractions. Although the mechanisms leading to repeat shortening are complex, understanding them can help researchers develop more precise therapeutic strategies with greater control of the editing process.

cell biology↗

bioRxiv2025

Directed evolution of compact RNA-guided nucleases for homology-directed repair in mammalian cells

RNA-guided nucleases enable DNA editing and offer promise for treating genetic diseases, particularly when used for precise sequence replacement. However, many of the most effective enzymes, such as S. pyogenes Cas9, are too large for delivery using vectors like adeno-associated virus (AAV). This has prompted interest in smaller alternatives from the Cas12f and TnpB families. Yet, these nucleases often show low activity in mammalian cells, limiting their utility. To address this, we used directed evolution in human cells to select variants with greatly improved activity. The resulting variants, Cas12f1Super and TnpBSuper, exhibited up to 11-fold increase in editing efficiency without increased off-target effects. When tested as a base editor, Cas12f1Super showed up to 10-fold improvement relative to the previously engineered CasMINI, suggesting utility beyond nuclease-related activities. These compact and efficient genome editors expand the current toolkit and hold promise for both research and therapeutic use in mammalian systems.

bioengineering↗

bioRxiv2025

Cell-stereotyped DNA repair outcomes are widespread during genome editing

Genome editing outcomes are governed by DNA repair pathways that vary with cell type and state. We developed scOUT-seq (single-cell Outcomes Using Transcript sequencing), a scalable approach that jointly profiles transcriptomes and matched multi-allelic editing outcomes ranging from homology directed repair (HDR) to inter-chromosomal translocations. We mapped editing outcomes in human CD34 hematopoietic stem and progenitor cells (HSPCs), mouse LSK HSPC equivalents, human upper airway organoids, and mouse multi-organ in vivo editing. Profiling 500,000 alleles across 74 cell types, scOUT-seq revealed that outcomes in most cell subtypes differ markedly from the bulk average. Various cell types shifted major repair classes, preferred different molecular sequences, and even enriched large structural variants, with distinctive patterns of allelic co-occurrence. Surprisingly, rare stem subtypes diverged from prevalent progenitors, and inhibitory neuron subtypes efficiently incorporated HDR alleles. These data suggest the potential for tailored therapeutic editing that may have been missed by bulk measurements.

genomics↗

bioRxiv2025

Disruption of nucleolar integrity triggers cellular quiescence through organelle rewiring and secretion

The nucleolus is the largest membraneless nuclear organelle and a critical regulator of growth and stress responses, comprised of over 600 proteins involved in ribosome biogenesis. However, how the nucleolar function and architecture coordinate cell-wide adaptive programs remains unclear. Here, we combined multi-omics profiling with functional genomics to define the cellular consequences of nucleolar disruption. We identify a stress response pathway connecting the nucleolus to the Golgi apparatus, endo-lysosomal trafficking, and cellular secretion (NuGETS). Chronic nucleolar defects activate a TP53-dependent transcriptional program that promotes organelle expansion, enhances secretory activity, and induces cell cycle exit with prolonged quiescence and partial epithelial-to-mesenchymal transition (pEMT) phenotypes. A genome-wide CRISPRi screen uncovered 400 regulators of secretory and quiescent states upon nucleolar stress. These pathways are enriched in cancer progression associated with ribosomopathies. Our findings therefore link nucleolar stress responses and cancer development, revealing cell-wide regulatory mechanisms that safeguard survival when nucleolar function is compromised.

cell biology↗

bioRxiv2025

WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2

Inhibitors of the protein kinase WEE1 have emerged as promising agents for cancer therapy. In this study, we uncover synergistic interactions between WEE1 small- molecule inhibitors and defects in mRNA translation, mediated by activation of the integrated stress response (ISR) through the kinase GCN2. Using a pooled CRISPRi screen, we identify GSPT1 and ALKBH8 as factors whose depletion confer hypersensitivity to the WEE1 inhibitor, AZD1775. We demonstrate that this synergy depends on ISR activation, which is induced by the off-target activity of WEE1 inhibitors. Furthermore, PROTAC-based WEE1 inhibitors and molecular glues show reduced or no ISR activation, suggesting potential strategies to minimise off-target toxicity. Our findings reveal that certain WEE1 inhibitors elicit dual toxicity via ISR activation and genotoxic stress, with ISR activation being independent of WEE1 itself or cell-cycle status. This dual mechanism highlights opportunities for combination therapies, such as pairing WEE1 inhibitors with agents targeting the mRNA translation machinery. This study also underscores the need for more precise WEE1 targeting strategies to mitigate off-target effects, with implications for optimising the therapeutic potential of WEE1 inhibitors.

cancer biology↗

bioRxiv2025

Proximity determines donor candidacy during DNA double-stranded break homology directed repair

DNA double-stranded breaks (DSBs) are especially toxic events that can be reversed by homology-directed repair (HDR), wherein information is copied from an intact template molecule. RAD51 mediates initial DSB/template pairing during homology search. A major challenge in understanding homology search in cells is the lack of tools to monitor this process. We developed RAD51 proximity identification sequencing (RaPID-seq), a sensitive method that marks all candidate templates searched by RAD51. We find that HDR is hierarchical, such that DSB proximity determines template candidacy and subsequent recombination is unlocked by DSB/template homology. Sequences that lie outside the proximal window are not efficiently searched, even if identical in sequence. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.

molecular biology↗

bioRxiv2024

Deciphering regulation of FOXP3 expression in human conventional T cells

FOXP3 is a lineage-defining transcription factor (TF) for immune-suppressive regulatory T cells (Tregs). While mice exclusively express FOXP3 in Tregs, humans also transiently express FOXP3 in stimulated conventional CD4+ T cells (Tconvs). Mechanisms governing these distinct expression patterns remain unknown. Here, we performed CRISPR screens tiling the FOXP3 locus and targeting TFs in human Tregs and Tconvs to discover cis-regulatory elements (CREs) and trans-regulators of FOXP3. Tconv FOXP3 expression depended on a subset of Treg CREs and Tconv-selective positive (TcNS+) and negative (TcNS-) CREs. The CREs are occupied and regulated by TFs we identified as critical regulators of FOXP3. Finally, mutagenesis of murine TcNS- revealed that it is critical for restriction of FOXP3 expression to Tregs. We discover CRE and TF circuitry controlling FOXP3 expression and reveal evolution of mechanisms regulating a gene indispensable to immune homeostasis. HighlightsO_LIComprehensive CRISPR maps of CREs and TFs controlling FOXP3 in human Tregs and Tconvs C_LIO_LIKey TFs that control FOXP3 directly occupy and regulate CREs forming TF-CRE circuits C_LIO_LIA previously unknown negative CRE stringently restricts FOXP3 to Tregs in mice C_LI

immunology↗

bioRxiv2024

SP110 sequestration of SP100 protects against toxic filaments during innate immune signaling

Stimulation of the innate immune system by foreign RNA elicits a potent response against invading pathogens and can trigger cell death. The mechanisms by which cells balance a robust response with cell-intrinsic lethality are still being uncovered. Employing genome-wide CRISPR-Cas9 genetic screens with triphosphorylated RNA stimulation, we identify speckled protein 110 (SP110) as a potent negative regulator of type 1 interferon-driven cell death. Death suppression by SP110 counteracts a death-promoting activity of another speckled protein, SP100. Both SP110 suppression and SP100 toxicity are mediated by direct interactions between the caspase activation and recruitment domains (CARDs) in each protein. SP100-induced death is mediated by homomeric CARD filaments that are disassembled by a heteromeric CARD interaction with SP110. Overexpression of SP100 is sufficient to overwhelm normal levels of SP110, leading to genotoxicity. Using cryo-EM and AlphaFold modeling, we develop and validate an atomic description of SP100 CARD filament formation and filament breaking by SP110. Genome-wide binding studies reveal that SP110 and SP100 normally associate at active promoters, but disruption of the CARD interaction releases SP100 to form toxic filaments. Overall, we uncover a novel regulatory partnership in human innate immunity that balances signal potency with cell intrinsic lethality.

immunology↗

bioRxiv2023

CCAR1 promotes DNA repair via an unanticipated role in alternative splicing

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology directed repair (HDR) in the context of [~]18000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi Anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

molecular biology↗

bioRxiv2023

Comprehensive Interrogation of Synthetic Relationships in the Human DNA Damage Response

The DNA damage response (DDR) is a multi-faceted network of pathways that preserves genome stability. Unraveling the complementary interplay between these pathways remains a challenge. Here, we comprehensively mapped genetic interactions for all core DDR genes using combinatorial CRISPRi screening. We discovered myriad new connections, including interactions between cancer genes and small molecule targets. We focused on two of the strongest interactions: FEN1/LIG1:WDR48 and FANCM:SMARCAL1. First, we found that WDR48 works with USP1 to restrain overactive translesion synthesis in FEN1/LIG1-deficient cells, and that a preclinical inhibitor of USP1 specifically kills FEN1-deficient cells. Second, we found that SMARCAL1 and FANCM suppress DNA double-strand break (DSB) formation at TA-rich repeats in late replicating regions that otherwise escape into mitosis and cause nuclear fragmentation. We present fundamental insights into genome maintenance processes and our dataset provides a springboard for mechanistic investigations into connections between DDR factors and suggests multiple interactions that could be exploited in cancer therapy.

cell biology↗

bioRxiv2023

Epitope Engineered Human Haematopoietic Stem Cells are Shielded from CD123-targeted Immunotherapy

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE) or chimeric antigen receptor (CAR) cells is very effective for haematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for immunotherapy of myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various haematological malignancies, including acute myeloid leukaemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). However, shared expression of CD123 on healthy haematopoietic stem and progenitor cells (HSPCs) bears the risk for extensive myelotoxicity upon targeted depletion. Here, we demonstrate that rationally designed, epitope-engineered HSPCs were completely shielded from CD123-targeted immunotherapy but remained fully functional while CD123-deficient HSPCs displayed a competitive disadvantage. Thus, molecularly shielded HSPCs could allow tumor-selective targeted immunotherapy and in parallel enable rebuilding a fully functional haematopoietic system. We envision that this approach is broadly applicable to many targets and cells, could render hitherto undruggable targets accessible to immunotherapy and will allow continued posttransplant immunotherapy, for instance to treat minimal residual disease (MRD) or be used as a salvage therapy. Since the function of the engineered targets is preserved, multiplexed molecular shielding could also enable targeted combination immunotherapies to address tumor heterogeneity. More generally, epitope shielding will be applicable for replacement of other cell types including the many immune cells which are currently being considered for engineered cellular therapies.

bioengineering↗

bioRxiv2023

C-terminal amides mark proteins for degradation via SCF/FBXO31

During normal cellular homeostasis unfolded and mis-localized proteins are recognized and removed, preventing the build-up of toxic byproducts1. When protein homeostasis is perturbed during aging, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites2, 3. Such modifications have been proposed to trigger the selective removal of chemically marked proteins3-6; however, discovering modifications sufficient to induce protein degradation has remained challenging. Using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified the SCF/FBXO31 ubiquitin ligase as a reader of C-terminal amides, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed following oxidative stress. A dominant human mutation found in neurodevelopmental disorders switches CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins.

cell biology↗

bioRxiv2023

BCAT1 inhibition affects CD8+ T cell activation, exhaustion, and tumoral immunity by altering iron homeostasis

The present study explores the role of the cytosolic branched chain amino acid aminotransferase (BCAT1) in CD8+ T cell activation, in general, and tumor immunity, in particular, and identifies a non-canonical function of the protein in iron homeostasis. Pharmacologic inhibition of BCAT1 using the novel drug ERG245 abrogates the effector functions of CD8+ T cells in vitro and metabolically reprograms the cells towards increased OXPHOS. In vivo, it suppresses activation of CD8+ T cells in DSS colitis leading to improved disease outcomes. Remarkably, withdrawal of BCAT1 inhibition further amplifies OXPHOS and gives rise to CD8+ T cells with increased cytotoxicity in vitro and in vivo. When combined with an anti-PD-1 treatment, temporal BCAT1 inhibition dramatically increases anti-PD-1 efficacy inducing complete and durable tumor regressions in the moderately immunogenic CT26 tumor model. Single cell RNA-seq data link expression of Bcat genes to exhausted T cells within the tumor microenvironment of human cancer patients, whereas in vitro assays indicate that BCAT1 inhibition partially prevents the adoption of a terminally exhausted phenotype by CD8+ T cells. We propose BCAT1 as a target for cancer combinatory therapies. SIGNIFICANCEThe study explores for the first time the role of BCAT1 in CD8+ T cell activation and proposes novel strategies for using BCAT1 inhibitors in cancer and beyond. It demonstrates that BCAT1 exerts its function without significantly altering branched chain amino acid (BCAA) levels through a mechanism that controls iron homeostasis, a novel non-canonical mechanism of action, and implicates BCAT1 in the adoption of an exhausted phenotype by T cells found in human cancers. While the majority of metabolic drugs temper OXPHOS, it demonstrates that an agent that increases OXPHOS in CD8+ T cells can be used successfully as an immune-oncology drug.

immunology↗

bioRxiv2022

Engineering of the Endogenous HBD promoter increases HbA2

The {beta}-hemoglobinopathies, such as sickle cell disease and {beta}-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of {beta}-globin subunits in adult hemoglobin. Many gene editing efforts to treat the {beta}-hemoglobinopathies attempt to correct {beta}-globin mutations or increase {gamma}-globin for fetal hemoglobin production. {delta}-globin, the subunit of adult hemoglobin A2, has high homology to {beta}-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of {delta}-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of {delta}-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate {delta}-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of {delta}-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total {beta}-like globins. Edited CD34+ hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 35% HBD. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat {beta}-hemoglobinopathies.

genetics↗

bioRxiv2022

TREX1 restricts CRISPR-Cas9 genome editing in human cells

CRISPR-Cas mediated homology-directed repair (HDR) can flexibly introduce desired mutations at targeted sites in a genome. But achieving high HDR efficiencies is a major hurdle in many cellular contexts. Moreover, cells from patients with mutations in DNA repair factors can exhibit low CRISPR-Cas-mediated HDR, complicating genome editing as a potential treatment. We used genome-wide screening in Fanconi anemia (FA) patient lymphoblastic cell lines to uncover suppressors of CRISPR-Cas mediated HDR. Surprisingly, we found that a single exonuclease called TREX1 is an important determinant of HDR efficiency when single-stranded templates are used as a repair template. TREX1 expression acts as a biomarker for CRISPR-Cas mediated HDR, such that cell lines expressing high levels of TREX1 have poor HDR that can be rescued by TREX1 removal. CRISPR-Cas mediated HDR can also be rescued by using single-stranded DNA templates that are chemically protected in a manner consistent with TREX1s exonucleolytic activity. Overall, our data provide a mechanistic explanation for why some cells are easier to edit than others and suggest a route to increase CRISPR-Cas mediated HDR in TREX-expressing context.

molecular biology↗

bioRxiv2022

Human ATG3 contains a non-canonical LIR motif crucial for its enzymatic activity in autophagy

Macroautophagy is one of two major degradation systems in eukaryotic cells. Regulation and control of autophagy is often achieved through the presence of short peptide sequences called LC3 interacting regions (LIR) in autophagy-involved proteins. Using a combination of new protein-derived activity-based probes, protein modelling and X-ray crystallography, we identified a non-canonical LIR motif in the human E2 enzyme responsible for LC3 lipidation, ATG3. The LIR motif is present in the flexible region of ATG3 and adopts an uncommon {beta}-sheet structure binding to the backside of LC3. We show that the {beta}-sheet conformation is crucial for its interaction with LC3. In cellulo studies provide evidence that LIRATG3 is required for LC3 lipidation and ATG3[~]LC3 thioester formation. Removal of LIRATG3 negatively impacts the rate of thioester transfer from ATG7 to ATG3. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/502437v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@f952e4org.highwire.dtl.DTLVardef@b0fe6aorg.highwire.dtl.DTLVardef@eeabcorg.highwire.dtl.DTLVardef@154ee1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Adenine base editing is an efficient approach to restore function in FA patient cells without double-stranded DNA breaks

Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions lead to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from an FA patient was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.

molecular biology↗