Biology subjects

Hart, T.

Publications and source records attributed to Hart, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Combinatorial CRISPR/Cas12a excises targeted loci in the mammalian genome at very low efficiency

The CRISPR/Cas12a system has emerged as a highly useful tool for genomic editing due to its ability to process multiple guide RNAs (gRNAs) from a single promoter. In this study, we explored the potential of combinatorial CRISPR/Cas12a to excise targeted loci in the mammalian genome by inducing double-strand breaks (DSBs) upstream and downstream of targeted exons. We designed a library of gRNA pairs targeting introns flanking 54 exons of 18 essential genes and 17 exons of 10 nonessential genes, aiming to assess the efficiency of Cas12 in excising targeted loci. While single gRNAs directly targeting essential exons produced expected fitness defects in cells, pairs of guides targeting flanking intronic regions resulted in only trivial viability effects, suggesting that excision rarely occurred. Further analysis showed no meaningful correlation between gRNA efficiency, orientation, GC content, or cut site geometry and the success of locus excision. These findings indicate that, under the conditions tested, the Cas12a system does not yield locus excision at the efficiency required for sensitive negative selection assays.

systems biology↗

bioRxiv2026

Mapping the genetic landscape of the DNA damage response with Cas12a-based combinatorial knockout screens

The DNA damage response (DDR) is a complex network of cellular pathways that ensures the faithful maintenance of our genomes upon a wide array of genomic insults. To elucidate the functional architecture of this network, we conducted unbiased genetic interaction screens using the Cas12a genome editor to disrupt 233 DDR genes frequently mutated in cancer and other genetic diseases, either individually or in pairwise combinations. This approach enabled us to assess the phenotypic effects induced by the disruption of >27,000 DDR gene pair combinations under unperturbed cell growth conditions. From this analysis, we identified over 750 high-confidence positive (buffering) or negative (synthetic lethal/sick) gene-gene interactions, along with multiple connections between previously unlinked DDR pathways and modules, allowing us to define novel aspects of the cellular response to spontaneous, DNA replication-associated DNA damage. Among the identified genetic interactions, we uncovered profound synthetic lethal interactions between genes encoding 1) the translesion polymerase REV1-Pol {zeta} complex and the MCM8-MCM9-HROB DNA helicase complex; 2) Fanconi Anemia (FA) proteins and the mitotic DNA repair factors GEN1, CIP2A, and RHINO; and 3) the DNA translocase SMARCAL1 and components of the FANCM complex, suggesting novel opportunities for targeted therapies in tumors carrying mutations in these genes. Additionally, we identified robust suppressor interactions between the DCLRE1B gene encoding the nuclease APOLLO and the core non-homologous end joining (NHEJ) genes XRCC4, LIG4, and NHEJ1, suggesting that NHEJ impairs the fitness of APOLLO-deficient cells. This work provides a functional map of the DDR network and demonstrates the power of Cas12a-based screens for identifying synthetic lethal and buffering interactions with therapeutic potential.

genetics↗

bioRxiv2026

AN UPDATED POPULATION ESTIMATE FOR NORTHERN GANNETS ACROSS THEIR NORTH-EAST ATLANTIC BREEDING RANGE FOLLOWING THE 2022 OUTBREAK OF HIGH PATHOGENICITY AVIAN INFLUENZA

Northern gannets (Morus bassanus) have been regarded as a seabird success story, due to population increases throughout the 20th and 21st century contrasting with global seabird declines. However, in 2022 gannets experienced a severe outbreak of High Pathogenicity Avian Influenza (HPAI) across their global distribution, leading to an urgent need to reassess their population status. This study presents breeding gannet census numbers for 2023/24 from all colonies across the North-East Atlantic metapopulation (Great Britain, Ireland, the Channel Islands, Iceland, Norway, the Faroe Islands, France, Germany, Russia). Gannet numbers decreased by 17% across the North-East Atlantic metapopulation between the 2013/14 and 2023/24 census from 414,598 to 345,854 apparently occupied sites (AOS), a global decrease of at least 13%. The bulk of the reduction in AOS was driven by the largest colonies (>10,000 AOS) each losing tens of thousands of AOS. These figures likely underestimate the impact of the HPAI outbreak worldwide, since most colonies will have increased between the last census in 2013/14 and the 2022 HPAI outbreak, and the Canadian breeding population was last counted pre-HPAI outbreak. Scotland still holds the largest proportion of both the North-East Atlantic metapopulation (59%), and the world population (46%), while Great Britain, Ireland and the Channel Islands together hold 83% of the North-East Atlantic metapopulation and 64% of the world population. This study not only presents an updated population census for gannets in the North-East Atlantic but illustrates the large-scale impacts of a disease outbreak on a seabird species across its global range and highlights the importance of more regular census efforts to better quantify the demographic consequences of such events.

ecology↗

bioRxiv2026

Analysis of Combinatorial Knockout CRISPR screens with GRAPE: Genetic interaction Regression Analysis of Pairwise Effects

Genetic interactions (GI) reveal functional relationships for understanding gene function and identifying candidate therapeutic vulnerabilities. Combinatorial CRISPR technologies enable genome-scale GI mapping in mammalian cells, but existing analytical methods lack systematic validation against ground truths. We introduce GRAPE (Genetic interaction Regression Analysis of Pairwise Effects), a computational framework that identifies GIs from pooled CRISPR screens by using linear regression to estimate single-gene phenotypes and detecting deviations from expected double-knockout effects. To enable rigorous benchmarking, we developed Synulator, a pipeline that simulates realistic CRISPR screen data with defined synthetic lethal interactions while preserving experimental noise profiles. In simulated screens, GRAPE achieves greater precision and recall compared to existing methods, particularly for interactions with weaker effect sizes. Applying GRAPE to published combinatorial screens across cell lines and CRISPR platforms demonstrates concordance with original findings while identifying additional high-confidence interactions. GRAPE provides a robust, versatile tool for GI mapping, advancing functional genomics and the systematic discovery of synthetic lethal targets in cancer. TeaserA regression-based framework and simulations enable accurate detection of GIs from combinatorial CRISPR screens.

systems biology↗

bioRxiv2026

Sequential, Chromosome-Specific Glutamine Synthetase Double Knockout with Cas-CLOVER Establishes Enhanced CHO Platforms for Cell Line Development

Cas-CLOVER is an emerging high-fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas-CLOVER to establish a robust, gene-edited platform in suspension-adapted CHO-K1 cells supporting cell line development (CLD) for biopharmaceutical production. An attractive strategy for high yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1). To compare editing efficiency, we evaluated Cas-CLOVER and Cas9 at both GS loci using the Neon Transfection System. Cas-CLOVER achieved 84% editing at GS5 and 74% at GS1, markedly higher than Cas 9. Leveraging Cas-CLOVERs dual-guide RNA design, we generated a GS5 single knockout (GS5-SKO) and subsequently a double knockout (GS-DKO) line at both the GS5 and GS1 loci, both with none detected off-target mutations analyzed in 40 predictably off-target sites. For functional validation, these cell lines were engineered with the proprietary Harbor-IN transposase system to stably express trastuzumab. Using an optimized protocol, the resulting GS-DKO platform, termed CleanCut GS CHO, enabled stringent selection and yielded high-producing clones with cell-specific productivity exceeding 100 pg/cell/day and antibody titers greater than 5 g/L in 24 deep well-plate fed-batch cultures after 14 days. The antibody titer stability analysis showed consistency over 60 generations. Collectively, these findings establish Cas-CLOVER as a versatile genome editing tool for developing high-yield CHO host platforms in CLD.

bioengineering↗

bioRxiv2025

Functional modules predict cancer-relevant genetic interactions in mammalian cells

Genetic interactions can reveal gene function and identify disease-relevant synthetic lethals, but resource-intensive screening in human cells constrains experimental scope and limits our understanding of how generalizable sparse hits are. Here, we leverage principles from yeast genetic networks to identify human gene modules involved in receptor tyrosine kinase signaling and DNA damage response that are predicted to be enriched for genetic interactions, then use our enCas12a-based In4mer combinatorial knockout platform to efficiently screen these modules across diverse cancer cell lines. We identify hundreds of unreported synthetic lethals, including a dense network within the protein N-linked glycosylation machinery that shows both evolutionary conservation and network rewiring, and we confirm that interactions discovered in 2D cell culture are maintained in more physiologically relevant models. Critically, a meta-analysis within and between screens highlights sources of variation in published results, reveals the potential for false positives in screens with many essential genes, and demonstrates the need to move beyond DDR for actionable synthetic lethalities.

systems biology↗

bioRxiv2025

Mouse Models Uniformly Featuring Human-like Lesions Harboring Drug-tolerant Mycobacterium tuberculosis

Mouse models have been key to studies of tuberculosis pathogenesis and drug efficacy, but many, such as those employing BALB/c mice, fail to reproduce the full range of heterogenous microniches observed in well-structured human lesions, which feature hypoxic caseous cores of necrotic debris surrounded by infected foamy macrophages. The granuloma presents a variety of environments differing in levels of oxygen, ions, nutrients, and intra versus extracellular residence, which determine the physiological state of the infecting bacillus and its susceptibility to immune or drug control. Recently, alternative mouse strains such as C3HeB/FeJ have allowed the study of infection and treatment in the context of these varied environments but exhibit substantial inconsistency in development of human-like lesions, both within and between individual mice. Building on the observation that inducible nitric oxide synthase (Nos2)-deficient mice consistently develop hypoxic necrotic lesions, we have established two simplified models with infection by the aerosol route. The first uses the slightly attenuated M. tuberculosis R1Rv strain, which produces a progressive infection that is contained at a high stable burden by an adaptive immune response. In the second model, vaccination with the attenuated {Delta}RD1, pantothenate auxotroph mc2 6230 protects from an otherwise lethal infection with virulent M. tuberculosis Erdman. This model reflects most contemporary tuberculosis infections, which take place in the context of a pre-existing immune response from vaccination. Both variations uniformly develop well-structured hypoxic necrotic lesions harboring drug tolerant bacteria. These refined models will be useful in studies of M. tuberculosis infection and treatment.

microbiology↗

bioRxiv2025

Integrated Metabolic Complex Genetic Interaction 1 Network of Chromosome 4p Loss in Basal Breast Cancer

Basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and exhibits a recurrent large chromosomal deletion in chromosome 4p (chr4p). Chr4p loss is associated with poor survival, evolves early in tumorigenesis and confers on cells a proliferative state. Here, we map the integrated metabolic complex genetic interaction network of chr4p in basal breast cancer to identify targetable vulnerabilities. Differential gene expression analysis of patient derived xenografts and cancer cell models revealed that chr4p loss is associated with changes in cellular energetics and reduction/oxidation balance. Analysis of DepMap pooled genome-wide CRISPR-Cas9 screens identified complex genetic interactions specific to chr4p deletion in basal breast cancer cell models. Functional assays revealed that chr4p loss is associated with disrupted mitochondrial respiratory function and reduced glycolytic capacity, suggesting metabolic rewiring. Increased reactive oxygen species and lipid peroxidation compromised antioxidant defense mechanisms. Ultimately, this study sheds light on targeted therapies for basal breast cancer harboring large chromosomal deletions.

genomics↗

bioRxiv2025

Double-CRISPR Knockout Simulation (DKOsim): A Monte-Carlo Randomization System to Model Cell Growth Behavior and Infer the Optimal Library Design for Growth-Based Double Knockout Screens

Advances in functional genomic technology, notably CRISPR using Cas9 or Cas12, now allow for large-scale double perturbation screens in which pairs of genes are inactivated, allowing for the experimental detection of genetic interactions (GIs). However, as it is not possible to validate GIs in high-throughput, there is no gold standard dataset where true interactions are known. Hence, we constructed a Double-CRISPR Knockout Simulation (DKOsim), which allows users to reproducibly generate synthetic simulation data where the single gene fitness effect of each gene and the interaction of each gene pair can be specified by the investigator. We adapted Monte-Carlo randomization methods to extend single knockout simulation methods to double knockout designs, which simulate the gene-gene interactions between all possible combinations of the input genes. Using DKOsim, we generated simulated datasets that closely resemble real double knockout CRISPR datasets in terms of Log Fold Change (LFC), GI distribution, and replicate correlation. We further inferred optimal CRISPR library designs by systematically investigating critical experimental parameters including depth of coverage, guide efficiency, and the variance of initial guide distribution. This simulation scheme will help to identify optimal computational methods for GI detection and aid in the design of future dual knockout CRISPR screens. Author SummaryWe designed DKOsim to simulate CRISPR double knockout screens by modeling cell division behavior with both single knockout (SKO) and double knockout (DKO) constructs via Monte-Carlo randomization samplers. Running DKOsim at large scale, we identified the asymptotic tuning points that optimize genetic interaction (GI) identification performance by the delta-LFC (dLFC) method compared to the simulated truth. We show that DKOsim is tunable to approximate actual dual-CRISPR knockout screening data. Comparing replicate correlation from DKOsim with experimentally generated data, DKOsim can be tuned based on users desires to reproduce a similar level of randomness to that observed in variety CRISPR screening conditions.

systems biology↗

bioRxiv2025

Evidence of Escherichia coli Regulating Drosophila melanogaster Behavior

Chemosensory systems are critical for insect survival, enabling host seeking, food acquisition, and oviposition site selection. While insect-associated microbes are known to influence host development and immunity, their role in modulating chemosensory behavior remains poorly understood. Here, we demonstrate that Escherichia coli, a bacterium identified in non-gut tissues of Aedes albopictus and experimentally reintroduced into axenic Drosophila melanogaster, alters both larval and adult sensory-driven behaviors. In larvae, E. coli infection modified phototaxis and mechanosensory responses across genotypes, while tunneling and thermosensory behaviors were specifically dependent on the ionotropic receptor co-receptor IR25a. In adults, E. coli increased attraction to fermentation cues (apple cider vinegar, ethanol) and enhanced sucrose consumption in wild-type and Orco-deficient flies, but not in IR25a-deficient mutants. Gas chromatography-mass spectrometry revealed that E. coli shifted cuticular hydrocarbon composition toward shorter-chain alkanes and increased the sex pheromone 9-tricosene in an IR25a-dependent manner. Together, these findings show that E. coli broadly reprograms insect sensory behavior, with IR25a serving as a critical mediator of microbial influence on chemosensory and physiological traits. This work identifies a previously unrecognized role for E. coli in shaping insect behavior and chemical ecology, providing a foundation for investigating microbial contributions to host-microbe coevolution and for exploring microbial cues as novel, environmentally sustainable tools for insect control. ImportanceInsects depend on their sense of smell and taste to find food, mates, and safe places to reproduce. These behaviors drive the spread of insect-borne diseases and cause major agricultural losses worldwide. Our work shows that a common bacterium, Escherichia coli, can reshape insect sensory behaviors, influencing how flies respond to light, temperature, food, and even the chemical signals on their outer surface that control mating. Importantly, these effects are linked to a specific sensory receptor, IR25a, which is conserved across many insect species. This finding reveals a previously unknown role for microbes in fine-tuning insect behavior. Understanding how bacteria influence insect sensory systems not only provides insight into host-microbe interactions but also opens the door to new, environmentally friendly ways of managing insect pests by targeting their behavior rather than killing them outright.

microbiology↗

bioRxiv2024

Secretory leukocyte protease inhibitor influences periarticular joint inflammation in B. burgdorferi-infected mice

Lyme disease, caused by Borrelia burgdorferi, is the most common tick-borne infection in the United States. Arthritis is a major clinical manifestation of infection, and synovial tissue damage has been attributed to the excessive pro-inflammatory responses. The secretory leukocyte protease inhibitor (SLPI) promotes tissue repair and exerts anti-inflammatory effects. The role of SLPI in the development of Lyme arthritis in C57BL/6 mice, which can be infected with B. burgdorferi, but only develop mild joint inflammation, was therefore examined. SLPI-deficient C57BL/6 mice challenged with B. burgdorferi had a higher infection load in the tibiotarsal joints and marked periarticular swelling, compared to infected wild type control mice. The ankle joint tissues of B. burgdorferi-infected SLPI-deficient mice contained significantly higher percentages of infiltrating neutrophils and macrophages. B. burgdorferi-infected SLPI-deficient mice also exhibited elevated serum levels of IL-6, neutrophil elastase, and MMP-8. Moreover, using a recently developed BASEHIT (BActerial Selection to Elucidate Host-microbe Interactions in high Throughput) library, we found that SLPI directly interacts with B. burgdorferi. These data demonstrate the importance of SLPI in suppressing periarticular joint inflammation in Lyme disease.

microbiology↗

bioRxiv2024

Therapeutic modulation of ROCK overcomes metabolic adaptation of cancer cells to OXPHOS inhibition and drives synergistic anti-tumor activity

Genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A in non-small cell lung cancer. Previously, we and others have identified that SMARCA4-mutant lung cancers are highly dependent on oxidative phosphorylation (OXPHOS). Despite initial excitements, therapeutics targeting metabolic pathways such as OXPHOS have largely been disappointing due to rapid adaptation of cancer cells to inhibition of single metabolic enzymes or pathways, suggesting novel combination strategies to overcome adaptive responses are urgently needed. Here, we performed a functional genomics screen using CRISPR-Cas9 library targeting genes with available FDA approved therapeutics and identified ROCK1/2 as a top hit that sensitizes cancer cells to OXPHOS inhibition. We validate these results by orthogonal genetic and pharmacologic approaches by demonstrating that KD025 (Belumosudil), an FDA approved ROCK inhibitor, has highly synergistic anti-cancer activity in vitro and in vivo in combination with OXPHOS inhibition. Mechanistically, we showed that this combination induced a rapid, profound energetic stress and cell cycle arrest that was in part due to ROCK inhibition-mediated suppression of the adaptive increase in glycolysis normally seen by OXPHOS inhibition. Furthermore, we applied global phosphoproteomics and kinase-motif enrichment analysis to uncover a dynamic regulatory kinome upon combination of OXPHOS and ROCK inhibition. Importantly, we found converging phosphorylation-dependent regulatory cross-talk by AMPK and ROCK kinases on key RHO GTPase signaling/ROCK-dependent substrates such as PPP1R12A, NUMA1 and PKMYT1 that are known regulators of cell cycle progression. Taken together, our study identified ROCK kinases as critical mediators of metabolic adaptation of cancer cells to OXPHOS inhibition and provides a strong rationale for pursuing ROCK inhibitors as novel combination partners to OXPHOS inhibitors in cancer treatment.

cancer biology↗

bioRxiv2024

Optimal methods for analyzing targeted pairwise knockout screens

BackgroundSynthetic lethality offers a promising strategy for cancer treatment by targeting genetic vulnerabilities unique to tumor cells, leading to selective tumor cell death. However, single-gene knockout screens often miss functional redundancy due to paralog genes. Multiplex CRISPR systems, including various Cas9 and Cas12a platforms, have been developed to assay genetic interactions, yet no systematic comparison of method to identify synthetic lethality from CRISPR screens has been conducted. ResultsWe evaluated data from four in4mer CRISPR/Cas12a screens in cancer cell lines, using three bioinformatic approaches to identify synthetic lethal interactions: delta log fold change (dLFC), Z-transformed dLFC (ZdLFC), and rescaled dLFC (RdLFC). Both ZdLFC and RdLFC provided more consistent identification of synthetic lethal pairs across cell lines compared to the unscaled dLFC method. ConclusionsThe ZdLFC method offers a robust framework for scoring synthetic lethal interactions from paralog screens, providing consistent results across different cell lines without requiring a training set of known positive interactors.

systems biology↗

bioRxiv2024

Characterization of Collaborative Cross mouse founder strain CAST/EiJ as a novel model for lethal COVID-19

Mutations in SARS-CoV-2 variants of concern (VOCs) have expanded the viral host range beyond primates, and a limited range of other mammals, to mice, affording the opportunity to exploit genetically diverse mouse panels to model the broad range of responses to infection in patient populations. Here we surveyed responses to VOC infection in genetically diverse Collaborative Cross (CC) founder strains. Infection of wild-derived CC founder strains produced a broad range of viral burden, disease susceptibility and survival, whereas most other strains were resistant to disease despite measurable lung viral titers. In particular, CAST/EiJ, a wild-derived strain, developed high lung viral burdens, more severe lung pathology than seen in other CC strains, and a dysregulated cytokine profile resulting in morbidity and mortality. These inbred mouse strains may serve as a valuable platform to evaluate therapeutic countermeasures against severe COVID-19 and other coronavirus pandemics in the future.

immunology↗

bioRxiv2024

Metabolic Reprogramming by Mutant GNAS Creates an Actionable Dependency in Intraductal Papillary Mucinous Neoplasms of the Pancreas

ObjectiveOncogenic "hotspot" mutations of KRAS and GNAS are two major driver alterations in Intraductal Papillary Mucinous Neoplasms (IPMNs), which are bona fide precursors to pancreatic ductal adenocarcinoma. We previously reported that pancreas-specific KrasG12D and GnasR201C co-expression in p48Cre; KrasLSL-G12D; Rosa26LSL-rtTA; Tg (TetO-GnasR201C) mice ("Kras;Gnas" mice) caused development of cystic lesions recapitulating IPMNs. Here, we aim to unveil the consequences of mutant GnasR201C expression on phenotype, transcriptomic profile, and genomic dependencies. DesignWe performed multimodal transcriptional profiling (bulk RNA sequencing, single cell RNA sequencing, and spatial transcriptomics) in the "Kras;Gnas" autochthonous model and tumor-derived cell lines (Kras;Gnas cells), where GnasR201C expression is inducible. A genome-wide CRISPR/Cas9 screen was conducted to identify potential vulnerabilities in KrasG12D;GnasR201Cco-expressing cells. ResultsInduction of GnasR201C - and resulting G(s)alpha signaling - leads to the emergence of a gene signature of gastric (pyloric type) metaplasia in pancreatic neoplastic epithelial cells. CRISPR screening identified the synthetic essentiality of glycolysis-related genes Gpi1 and Slc2a1 in KrasG12D;GnasR201C co-expressing cells. Real-time metabolic analyses in Kras;Gnas cells and autochthonous Kras;Gnas model confirmed enhanced glycolysis upon GnasR201C induction. Induction of GnasR201C made KrasG12D expressing cells more dependent on glycolysis for their survival. Protein kinase A-dependent phosphorylation of the glycolytic intermediate enzyme PFKFB3 was a driver of increased glycolysis upon GnasR201C induction. ConclusionMultiple orthogonal approaches demonstrate that KrasG12D and GnasR201C co-expression results in a gene signature of gastric pyloric metaplasia and glycolytic dependency during IPMN pathogenesis. The observed metabolic reprogramming may provide a potential target for therapeutics and interception of IPMNs. SUMMARYO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIActivating "hotspot" mutations of KRAS and GNAS are found in a majority of Intraductal Papillary Mucinous Neoplasms (IPMNs). C_LIO_LIExpression of mutant KRAS and GNAS drives development of IPMN-like cystic lesions in the murine pancreas that eventually progress to pancreatic ductal adenocarcinoma (PDAC). C_LI What this study addsO_LIMutant GNAS and the resulting aberrant G(s)alpha signaling drives a transcriptional signature of gastric (pyloric type) metaplasia in IPMNs with mucin production. C_LIO_LIAberrant G(s)alpha signaling enhances glycolysis via protein kinase A-dependent phosphorylation of the glycolytic enzyme PFKFB3. C_LIO_LIEnhanced glycolysis in KRAS;GNAS-mutated IPMN cells is validated via multiple orthogonal approaches in vitro and in vivo and represents an actionable metabolic vulnerability. C_LI How this study might affect research, practice or policyO_LIThe present study provides mechanistic insight into how aberrant G(s)alpha signaling alters the biology of Kras-mutant pancreatic epithelial neoplasia through metaplastic and metabolic reprogramming. C_LIO_LITargeting glycolysis in IPMNs may represent both a therapeutic avenue as well as an opportunity for intercepting progression to invasive cancer. C_LI

cancer biology↗

bioRxiv2024

Pheromone representation in the ant antennal lobe changes with age

While the neural basis of age-related decline has been extensively studied (1-3), less is known about changes in neural function during the pre-senescent stages of adulthood. Adult neural plasticity is likely a key factor in social insect age polyethism, where individuals perform different tasks as they age and divide labor in an age-dependent manner (4-9). Primarily, workers transition from nursing to foraging tasks (5, 10), become more aggressive, and more readily display alarm behavior (11-16) as they get older. While it is unknown how these behavioral dynamics are neurally regulated, they could partially be generated by altered salience of behaviorally relevant stimuli (4, 6, 7). Here, we investigated how odor coding in the antennal lobe (AL) changes with age in the context of alarm pheromone communication in the clonal raider ant (Ooceraea biroi) (17). Similar to other social insects (11, 12, 16), older ants responded more rapidly to alarm pheromones, the chemical signals for danger. Using whole-AL calcium imaging (18), we then mapped odor representations for five general odorants and two alarm pheromones in young and old ants. Alarm pheromones were represented sparsely at all ages. However, alarm pheromone responses within individual glomeruli changed with age, either increasing or decreasing. Only two glomeruli became sensitized to alarm pheromones with age, while at the same time becoming desensitized to general odorants. Our results suggest that the heightened response to alarm pheromones in older ants occurs via increased sensitivity in these two core glomeruli, illustrating the importance of sensory modulation in social insect division of labor and age-associated behavioral plasticity.

neuroscience↗

bioRxiv2024

Unselfish meiotic drive maintains heterozygosity in a parthenogenetic ant

According to Mendels second law, chromosomes segregate randomly in meiosis. Non-random segregation is primarily known for cases of selfish meiotic drive in females, in which particular alleles bias their own transmission into the oocyte1,2. Here, we report a rare example of unselfish meiotic drive for crossover inheritance in the clonal raider ant, Ooceraea biroi. This species produces diploid offspring parthenogenetically via fusion of two haploid nuclei from the same meiosis3. This process should cause rapid genotypic degeneration due to loss of heterozygosity, which results if crossover recombination is followed by random (Mendelian) segregation of chromosomes4,5. However, by comparing whole genomes of mothers and daughters, we show that loss of heterozygosity is exceedingly rare, raising the possibility that crossovers are infrequent or absent in O. biroi meiosis. Using a combination of cytology and whole genome sequencing, we show that crossover recombination is, in fact, common, but that loss of heterozygosity is avoided because crossover products are faithfully co-inherited. This results from a programmed violation of Mendels law of segregation, such that crossover products segregate together rather than randomly. This discovery highlights an extreme example of cellular "memory" of crossovers, which could be a common yet cryptic feature of chromosomal segregation.

evolutionary biology↗

bioRxiv2023

Combined genome-scale fitness and paralog synthetic lethality screens with just 44k clones: the IN4MER CRISPR/Cas12a multiplex knockout platform

Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for combinatorial genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different approaches and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the Cas12a platform provides superior sensitivity and assay replicability. We demonstrate that Cas12a can independently target up to four genes from a single guide array, and we build on this knowledge by constructing a genome-scale library that expresses arrays of four guides per clone, a platform we call in4mer. Our genome-scale human library, with only 49k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than four thousand paralog pairs, triples, and quads. Proof of concept screens in four cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state-of-the-art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.

systems biology↗