Biology subjects

Wilson, R. C.

Publications and source records attributed to Wilson, R. C..

At least 19 recordsLinked to original sources

bioRxiv2026

CRISPR-dPCR: A unified multiplex digital PCR framework for quantitative characterization of targeted knock-in

Accurate characterization of CRISPR knock-in requires distinguishing site-specific integration from total donor abundance and donor-derived background while measuring editing at the target site. Here, we present CRISPR-dPCR, a modular multiplex digital PCR framework combining a locked nucleic acid-enhanced cut-site assay with junction-based detection and two orthogonal short-amplicon strategies: Poisson linkage and homology-arm subtraction. We evaluated assay modules across genomic loci, neuroblastoma models and primary human T cells, with selected assays compared on two dPCR platforms. In genotype-defined clones and mixtures with low residual donor, homology-arm subtraction most closely recovered expected knock-in abundance. Linkage provided highly specific evidence of target-cargo association without junction-spanning amplification, whereas junction assays supplied boundary-specific confirmation but were more sensitive to amplicon length and multiplexing. Five-channel panels integrated cut-site editing, donor abundance, copy-number normalization and site-specific knock-in in a single reaction. Residual donor DNA limited quantitative interpretation, particularly in early post-editing samples. Modular extensions quantified defined unintended outcomes, including chromosomal translocations. CRISPR-dPCR integrates complementary measurements into a scalable analytical framework for genome-editing research, cell and gene therapy product development and applications in longitudinal clinical monitoring of targeted integration and transgene persistence.

molecular biology↗

bioRxiv2026

Chemically modified CRISPR enzymes for multi-organ genome editing in vivo

Delivery remains the main obstacle to the development of in vivo genome editing therapies. CRISPR ribonucleoproteins confer high editing activity with transient exposure but lack intrinsic cell entry and targeting. Here we introduce PERCEPT, a delivery platform featuring reversible, covalent modification of CRISPR enzymes. PERCEPT enables modular installation of shielding polymers, amphiphilic delivery peptides and targeting ligands, allowing traceless cytosolic release of the native editor. A formulation incorporating an amphiphilic delivery peptide and the neuron-targeting ligand TET1 edited approximately 56% of striatal volume and 78% of neurons within edited regions after local striatal administration. In the R6/2 Huntington's disease model, PERCEPT mediated targeted editing of the mutant human HTT transgene, reducing mutant huntingtin aggregate burden and shifting local transcriptional programs away from inflammatory and injury-associated states. Tissue-adapted formulations edited 53% of Muller glia following intravitreal delivery, increased skeletal muscle reporter fluorescence tenfold versus unconjugated control following intramuscular injection, and enabled lung airway epithelial cell that persisted for three months following intranasal delivery. Rapid screening revealed that local anatomical and cellular barriers require distinct surface display for optimal editing. These results establish reversible chemical modification as a general strategy for adapting CRISPR enzyme delivery across multiple target tissues in vivo.

bioengineering↗

bioRxiv2026

CRISPR-Mediated Targeting of BRAF Oncogenes in Pediatric Low-Grade Glioma

SummaryA catch-all intronic guide RNA pair excises the KIAA1549--BRAF oncofusion across its major variants, with productive junction excision confirmed by gain-of-function PCR in patient-derived glioma cells. An allele-specific guide selectively disrupts BRAF V600E, in patient-derived pediatric low-grade glioma cells. Pediatric low-grade glioma (pLGG) is the most common brain tumor of childhood, accounting for 30--50% of all pediatric central nervous system malignancies1. The disease is almost universally driven by activating mutations in the BRAF serine/threonine kinase: a chromosomal tandem duplication generating the KIAA1549--BRAF oncofusion in approximately 70% of cases, or the BRAF V600E gain-of-function point mutation in approximately 15%2. Current targeted pharmacotherapies, including the RAF inhibitor tovorafenib, require continuous dosing, are not allele-specific, and carry risks of long-term toxicity in children. A one-time genomic intervention that permanently disables the oncogenic BRAF alteration while preserving wild-type BRAF signaling represents a compelling therapeutic alternative. In this study, we describe the design and experimental validation of allele-specific CRISPR guide RNAs targeting both the KIAA1549--BRAF oncofusion and the BRAF V600E point mutation. For the oncofusion, we developed a double-cut intronic excision strategy in which a guide RNA targeting KIAA1549 intron 14 is paired with a guide RNA targeting BRAF intron 11. Because the genomic breakpoints of all four major fusion variants (KB 16:9, 15:9, 16:11, and 15:11) fall within these introns, a single guide pair can address the full landscape of fusion heterogeneity in a single intervention. For BRAF V600E, we exploited a unique PAM sequence created by the pathogenic TBA transversion at codon 600, enabling allele-specific SpCas9 and AsCas12a guide designs that distinguish the mutant from the wild-type allele at single-nucleotide resolution. We screened guide RNA candidates by ribonucleoprotein (RNP) nucleofection in A375 human melanoma cells (BRAF V600E homozygous) and in patient-derived 3635 PXA glioma cells (BRAF V600E heterozygous). The top KIAA1549 intron 14 guide, K9_i14_A_Cas9, achieved 66% indel frequency in A375 cells. The top BRAF intron 11 guides, B_i11_A_Cas9 and B_i11_D_Cas9, achieved 84% and 85% indel frequency, respectively. For BRAF V600E, the best allele-specific SpCas9 guide achieved l57% editing in A375 cells and l74% editing in 3635 PXA patient-derived glioma cells. Dual-cut excision of the KIAA1549--BRAF junction was confirmed by a gain-of-function PCR assay designed to detect the excision junction amplicon ([~]191 bp) produced by NHEJ-mediated rejoining of the KIAA1549 intron 14 and BRAF intron 11 cut ends.

cancer biology↗

bioRxiv2026

Modeling the journey as well as the destination: a control theory account of rotational navigation

Navigation requires estimating heading and transforming these estimates into actions. Prior models explain how self-motion and landmark cues are combined into heading estimates, but less is known about how these estimates are iteratively transformed into motor commands to reach a goal. Here, we hypothesized that navigation operates as a closed-loop process in which ongoing movement is updated by sensory prediction errors. To test this hypothesis, participants performed a goal-directed rotation task in virtual reality. On select trials, visual landmarks were shifted during movement, inducing a prediction error between the heading expected from self-motion estimates and the heading observed from the shifted landmarks. In parallel, we developed a closed-loop model of turning behavior that represents heading and angular velocity as jointly estimated states over time. This model accounts not only for final position--the destination--but also for the movement dynamics that produce it--the journey. The model predicts that landmark-induced visual prediction errors should produce rapid corrective changes in movement. Participant turning behavior qualitatively paralleled these model dynamics: acceleration changed after visual feedback, with larger landmark mismatches producing larger corrective responses. Together, these findings suggest that naturalistic movement depends on continuously transforming heading estimates into motor command through closed-loop control.

neuroscience↗

bioRxiv2026

Illusory path configurations reveal age-related differences in egocentric pointing variability

A consistent finding across studies with older adults is that they typically perform worse at spatial memory tasks, particularly those conducted in virtual reality and involving novel environments, compared to young adults. While the underlying reasons for this difference remain unclear, some proposed hypotheses include differences in sensory cue integration and cue conflict resolution. Here, we tested older (n = 29) and young adults (n = 28) in immersive and walkable virtual reality using both correctly rendered and illusory hallways to test how visual cues (i.e., an intersection) and self-motion cues are integrated. In the illusory or false-intersection condition, we hypothesized that participants who walked an uncrossed path would merge two disconnected intersections, creating the illusion of a crossed path. The overall accuracy and pointing patterns were similar between young and older adults in both true- and false-intersection conditions. We did find, however, a significant age by condition interaction effect in egocentric pointing variability where older adults showed lower variability in the illusory condition and higher variability in the control condition. At the same time, older adults also drew worse maps for the control condition compared to young adults. However, the pointing error correlated with the accuracy of maps drawn regardless of age, suggesting that the pointing patterns shown by both age groups related to their underlying representations of the paths. Our findings are inconsistent with a global deficit in allocentric navigation or path integration and instead suggest that more subtle differences in strategy use might manifest with age.

neuroscience↗

bioRxiv2026

Epigenetic plasticity is associated with enhanced tolerance to low temperature stress in woodland strawberry

Low temperature stress causes significant damage to the strawberry plant. During cold stress, plants undergo morphological and physiological changes often regulated at the genetic and/or epigenetic levels. Some strawberry cultivars are more cold-hardy than others. Using the diploid woodland strawberry as a model, we analyzed the effects of cold acclimation on methylome and transcriptome dynamics in the crowns and leaves of three ecotypes with contrasting cold tolerance. Alta, which was the most cold-tolerant ecotype, exhibited the highest genetic and epigenetic plasticity in response to cold. CHH-context methylation dominated the differentially methylated regions (DMRs) with more hypomethylation in crowns and hypermethylation in leaves. CG methylation was enriched in gene bodies, while non-CG methylation was prevalent in upstream and downstream regions. Our study revealed that less than a quarter of differentially methylated genes (DMGs) showed changes in transcript accumulation levels. This finding indicates that universal cold response in Fragaria vesca, as reflected by gene expression, cannot be mechanistically attributed to DNA methylation. The majority of differentially expressed differentially methylated genes (DEDMGs) were ecotype- and tissue-specific. Enrichment analysis revealed that these genes were involved in pathways related to stress tolerance, such as carbohydrate metabolism, lipid metabolism, ATP hydrolysis, and cellular detoxification. Each ecotype responded to cold through mobilization of its own set of differentially expressed genes (DEGs), DMGs, and DEDMGs, and variation in expression and methylation patterns exhibited by Alta, FDP817, and NCGR1363 suggest that cold signaling processes and survival depend on the tissue, ecotype, and geographical origin of the plants exposed to cold stress. Therefore, this study highlights the potential of both genetic markers and epialleles as molecular markers for the development of cold-tolerant octoploid strawberry cultivars that are better suited for propagation in Nordic climates.

plant biology↗

bioRxiv2025

Human Strategy Adaptation in Reinforcement Learning Resembles Policy Gradient Ascent

A hallmark of intelligence is the ability to adapt behavior to changing environments, which requires adapting ones own learning strategies. This phenomenon is known as learning to learn or meta-learning. Although well established in humans and animals, a computational framework that characterizes how biological agents adapt their learning strategies through experience remains elusive. Here we posit that humans update their learning strategies online through a gradient-based meta-learning process, effectively optimizing how they learn. However, estimating how these strategies evolve over time remains a significant challenge since traditional cognitive models, such as reinforcement learning (RL), typically assume that agents use static strategies. To address this, we introduce DynamicRL, a method that leverages neural networks to estimate the evolution of an individuals RL strategy by tracking cognitive parameters such as learning rates over time. Across four human bandit tasks, DynamicRL consistently outperforms traditional RL models with fixed parameters in fitting behavior, confirming that humans adapt their RL strategies over time. RL parameters estimated by DynamicRL reveal trajectories that systematically increase the expected reward of the RL strategy. The parameter updates at each step resemble policy gradient ascent, and their optimality correlates with the strength of the gradient signal. Moreover, these RL parameters evolve more slowly than decision variables, supporting the hierarchical relationship between strategy learning and value learning. Our work provides a computational framework that expands the hypothesis space from understanding strategies to understanding strategy adaptation, bridging adaptive behavior in biological and artificial intelligence through meta-learning.

animal behavior and cognition↗

bioRxiv2025

No Effect of Chronic or Acute Pain on Working Memory in the Sternberg Task

Memories of pain can last a lifetime, preventing future injuries, but this comes at the expense of remembering other concurrent experiences. For acute pain this cost is outweighed by the benefits but when pain is chronic, pain memory benefits are limited and may even contribute to maintenance of the disorder. Here we investigated two hypotheses, (1) pain takes up slots in working memory or (2) pain induces arousal above optimum levels at high task difficulty, leading to a decrease in performance. To do this, we used the Sternberg Task of working memory, in which the participant plays repeated trials where they are shown different sets of numbers and then asked to identify whether a probe number was in the set or not. The Sternberg Task is ideal for testing the two hypotheses by looking at pain-related changes in accuracy and response time. There was a replication of the response time increase with set size, as well as the effect of older age on working memory and pain threshold. However, we saw no pain effect on either response time, accuracy, or the relationship between these parameters and set size with either chronic pain or an acute painful thermal stimulus. Together, this suggests that pain does not impair working memory in the Sternberg Task.

neuroscience↗

bioRxiv2025

Deep Learning Improves Parameter Estimation in Reinforcement Learning Models

AO_SCPLOWBSTRACTC_SCPLOWCognitive models are widely used in psychology and neuroscience to formulate and test hypotheses about cognitive processes. These processes are characterized by model parameters, which are then used for scientific inference. The reliability of scientific conclusions from cognitive modeling depends critically on the reliability of parameter estimation, yet estimating parameters remains a universal challenge particularly when data are too limited to constrain them. In such cases, multiple sets of parameters may explain the experimental data equally well within the same model, raising the question of which parameters are scientifically meaningful. We refer to this problem as parameter ambiguity. In this paper, we investigate parameter ambiguity in reinforcement learning under two optimization methods. We employ the de facto Nelder-Mead method (fminsearch) and a neural network trained to estimate parameters using a modern deep learning pipeline, which has seen limited application in cognitive modeling. Across ten decision-making datasets, we consistently find that the two methods produce substantially different parameter estimates despite achieving nearly identical fitting performance. To address this ambiguity, we introduce a systematic evaluation framework that goes beyond predictive accuracy to assess generalizability, robustness, identifiability, and test-retest reliability, thereby offering principled guidance on which parameter estimates should inform scientific inference. Applying this framework reveals that the neural network with a deep learning pipeline outperforms across these metrics. Our study establishes parameter ambiguity as an underappreciated challenge with significant implications for scientific replicability, highlighting that the choice of optimization method is a critical factor shaping scientific conclusions. We advocate for our multi-faceted evaluation approach to ensure reliable scientific inference and for broader integration of modern deep learning pipelines into cognitive modeling.

animal behavior and cognition↗

bioRxiv2025

The dynamics of explore-exploit decisions suggest a threshold mechanism for reduced random exploration in older adults

When faced with a choice between exploring an unknown option vs exploiting an option they know well, older adults explore less and exploit more than younger adults. Recent work has suggested that one cause of this age difference in exploration is a reduction in the extent to which older adults use "random exploration" - exploration driven by behavioral variability. Here we investigate potential mechanisms for this age-related difference in random exploration through the lens of a drift diffusion model (DDM) of the explore-exploit choice. In this model, random exploration can be modulated by two mechanisms - the fidelity with which information about the choice is represented in the brain, the "signal-to-noise ratio" (SNR), and the amount of information required to make a decision, the "decision threshold." Reduced random exploration in aging could be caused either by an increase in signal-to-noise ratio or an increase in decision threshold in older adults. By fitting the DDM to choices and response times in a sample of healthy younger and older adults, we found that older adults had a lower SNR and a higher threshold than younger adults. This suggests that reduced random exploration in aging is driven by higher response thresholds in older adults, which may compensate for the reduced signal-to-noise ratio with which decision information is represented in the brain. Author SummaryThe balance of deciding to explore the unknown, versus exploiting the well-known, changes with age. Compared to younger counterparts, healthy older adults have reduced random exploration, in which choices appear uninfluenced by the value of options. To investigate the mechanism of reduced random exploration, reaction time and choices between two slot machines in the Horizon Task were modelled using the drift diffusion model (DDM). In the DDM there is an accumulation of evidence over time, until a boundary threshold for either the explore or exploit option is crossed. The DDM used here can distinguish between two different drivers of random exploration, changes in the signal-to-noise ratio (SNR), with which reward information is represented, and changes in the threshold required to make a decision. We showed that reduced random exploration in older adults results from a higher decision threshold. Whilst older adults had a lower SNR than younger adults, which could lead to more mistakes, older adults actually performed slightly better in the Horizon task than younger adults. Together this suggests that the higher decision threshold could be a healthy aging adaptation, which is overcompensating for the less accurate choices that could result from a lower SNR alone.

neuroscience↗

bioRxiv2024

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.

bioengineering↗

bioRxiv2024

Semantic Influences on Object Detection: Drift Diffusion Modeling Provides Insights Regarding Mechanism

AbstractResearch shows that semantics, activated by words, impacts object detection. Skocypec & Peterson (2022) indexed object detection via correct reports of where figures lie in bipartite displays depicting familiar objects on one side of a border. They reported 2 studies with intermixed Valid and Invalid labels shown before test displays and a third, control, study. Valid labels denoted display objects. Invalid labels denoted unrelated objects in a different or the same superordinate-level category in studies 1 & 2, respectively. We used drift diffusion modeling (DDM) to elucidate the mechanisms of their results. DDM revealed that, following Valid labels, drift rate toward the correct decision increased, i.e., SNR increased. Following Invalid labels, SNR was lower only for upright displays in study 2. Thresholds were higher in studies 1 & 2 than in control. That more evidence must be accumulated from displays that follow labels implies that familiar object detection entails semantic activation. Threshold was even higher following Invalid labels in study 2, suggesting that more evidence from the display is needed to resolve within- category conflicts. These results support the view that semantic networks are engaged in object detection.

neuroscience↗

bioRxiv2024

Detection of pathogens and antimicrobial resistant genes from urine within 5 hours using Nanopore sequencing

PurposeUrinary Tract Infection (UTI) is a prevalent global health concern accounting for 1-3% of primary healthcare visits. The current methods for UTI diagnosis have a high turnaround time of 3-5 days for pathogen identification and susceptibility testing. This work is a proof-of-concept study aimed at determining the detection limit by establishing a culture and amplification-free DNA extraction methodology from spiked urine samples followed by real-time Nanopore sequencing and data analysis. MethodsThis study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Pathogens were spiked at two clinically significant concentrations of 103 and 105 CFU/ml. Three commercial DNA extraction kits were investigated based on the quantity of isolated DNA, average processing time, elution volume and the average cost incurred per extraction. The outperforming kit was used for direct DNA extraction and subsequent sequencing on MinION and Flongle flowcells. ResultsThe Blood and Tissue kit outperformed the other kits. All pathogens were identified at a concentration of 105 CFU/ml within ten minutes, and the corresponding AMR genes were detected within three hours of the sequencing start. The overall turnaround time including the DNA extraction and sequencing steps was five hours. Moreover, we also demonstrate that the identification of some pathogens and antibiotic-resistance genes was possible at a spike concentration of 103 CFU/mL. ConclusionThis study shows great promise toward reducing the time required for making an informed antibiotic administration from approximately 48 hours to five hours thereby reducing the number of empirical doses and saving lives.

microbiology↗

bioRxiv2023

Frontal-midline oscillations index the evolution of spatial memory during active navigation

Previous research has demonstrated that humans combine multiple sources of spatial information such as self-motion and landmark cues, while navigating through an environment. However, it is unclear whether this involves comparing multiple representations obtained from different sources during navigation (parallel hypothesis) or building a representation first based on self-motion cues and then combining with landmarks later (serial hypothesis). We tested these two hypotheses (parallel vs. serial) in an active navigation task using wireless mobile scalp EEG recordings. Participants walked through an immersive virtual hallway with or without conflicts between self-motion and landmarks (i.e., intersections) and pointed toward the starting position of the hallway. We employed the oscillatory signals recorded during mobile wireless scalp EEG as means of identifying when participant representations based on self-motion vs. landmark cues might have first emerged. We found that path segments, including intersections present early during navigation, were more strongly associated with later pointing error, regardless of when they appeared during encoding. We also found that there was sufficient information contained within the frontal-midline theta and posterior alpha oscillatory signals in the earliest segments of navigation involving intersections to decode condition (i.e., conflicting vs. not conflicting). Together, these findings suggest that intersections play a pivotal role in the early development of spatial representations, suggesting that memory representations for the geometry of walked paths likely develop early during navigation, in support of the parallel hypothesis.

neuroscience↗

bioRxiv2022

T-cell cellular stress and reticulocyte signatures, but not loss of naive T lymphocytes, characterize severe COVID-19 in older adults

In children and younger adults up to 39 years of age, SARS-CoV-2 usually elicits mild symptoms that resemble the common cold. Disease severity increases with age starting at 30 and reaches astounding mortality rates that are ~330 fold higher in persons above 85 years of age compared to those 18-39 years old. To understand age-specific immune pathobiology of COVID-19 we have analyzed soluble mediators, cellular phenotypes, and transcriptome from over 80 COVID-19 patients of varying ages and disease severity, carefully controlling for age as a variable. We found that reticulocyte numbers and peripheral blood transcriptional signatures robustly correlated with disease severity. By contrast, decreased numbers and proportion of naive T-cells, reported previously as a COVID-19 severity risk factor, were found to be general features of aging and not of COVID-19 severity, as they readily occurred in older participants experiencing only mild or no disease at all. Single-cell transcriptional signatures across age and severity groups showed that severe but not moderate/mild COVID-19 causes cell stress response in different T-cell populations, and some of that stress was unique to old severe participants, suggesting that in severe disease of older adults, these defenders of the organism may be disabled from performing immune protection. These findings shed new light on interactions between age and disease severity in COVID-19.

immunology↗

bioRxiv2021

Combination and competition between path integration and landmark navigation in the estimation of heading direction

Successful navigation requires the ability to compute ones location and heading from incoming multisensory information. Previous work has shown that this multisensory input comes in two forms: body-based idiothetic cues, from ones own rotations and translations, and visual allothetic cues, from the environment (usually visual landmarks). However, exactly how these two streams of information are integrated is unclear, with some models suggesting the body-based idiothetic and visual allothetic cues are combined, while others suggest they compete. In this paper we investigated the integration of body-based idiothetic and visual allothetic cues in the computation of heading using virtual reality. In our experiment, participants performed a series of body turns of up to 360 degrees in the dark with only a brief flash (300ms) of visual feedback en route. Because the environment was virtual, we had full control over the visual feedback and were able to vary the offset between this feedback and the true heading angle. By measuring the effect of the feedback offset on the angle participants turned, we were able to determine the extent to which they incorporated visual feedback as a function of the offset error. By further modeling this behavior we were able to quantify the computations people used. While there were considerable individual differences in performance on our task, with some participants mostly ignoring the visual feedback and others relying on it almost entirely, our modeling results suggest that almost all participants used the same strategy in which idiothetic and allothetic cues are combined when the mismatch between them is small, but compete when the mismatch is large. These findings suggest that participants update their estimate of heading using a hybrid strategy that mixes the combination and competition of cues. Author summarySuccessful navigation requires us to combine visual information about our environment with body-based cues about our own rotations and translations. In this work we investigated how these disparate sources of information work together to compute an estimate of heading. Using a novel virtual reality task we measured how humans integrate visual and body-based cues when there is mismatch between them -- that is, when the estimate of heading from visual information is different from body-based cues. By building computational models of different strategies, we reveal that humans use a hybrid strategy for integrating visual and body-based cues -- combining them when the mismatch between them is small and picking one or the other when the mismatch is large.

neuroscience↗

bioRxiv2021

Genetic mapping and identification of a QTL determining tolerance to freezing stress in Fragaria vesca L.

Extreme cold and frost cause significant stress to plants which can potentially be lethal. Low temperature freezing stress can cause significant and irreversible damage to plant cells and can induce physiological and metabolic changes that impact on growth and development. Low temperatures cause physiological responses including winter dormancy and autumn cold hardening in strawberry (Fragaria) species, and some diploid F. vesca accessions have been shown to have adapted to low-temperature stresses. To study the genetics of freezing tolerance, a F. vesca mapping population of 142 seedlings segregating for differential responses to freezing stress was raised. The progeny was mapped using Genotyping-by-Sequencing and a linkage map of 2,918 markers at 851 loci was resolved. The mapping population was phenotyped for freezing tolerance response under controlled and replicated laboratory conditions and subsequent quantitative trait loci analysis using interval mapping revealed a single significant quantitative trait locus on Fvb2 in the physical interval 10.6 Mb and 15.73 Mb on the F. vesca v4.0 genome sequence. This physical interval contained 896 predicted genes, several of which had putative roles associated with tolerance to abiotic stresses including freezing. Differential expression analysis of the 896 QTL-associated gene predictions in the leaves and crowns from Alta and NCGR1363 parental genotypes revealed genotype-specific changes in transcript accumulation in response to low temperature treatment as well as expression differences between genotypes prior to treatment for many of the genes. The putative roles, and significant interparental differential expression levels of several of the genes reported here identified them as good candidates for the control of the effects of freezing tolerance at the QTL identified in this investigation and the possible role of these candidate genes in response to freezing stress is discussed.

genetics↗

bioRxiv2020

Heterotrimeric PCNA Increases the Activity and Fidelity of Dbh, a Y-family Translesion DNA Polymerase Prone to Creating Single-Base Deletion Mutations

Dbh is a Y-family translesion DNA polymerase from Sulfolobus acidocaldarius, an archaeal species that grows in harsh environmental conditions. Biochemically, Dbh displays a distinctive mutational profile, creating single-base deletion mutations at extraordinarily high frequencies (up to 50%) in specific repeat sequences. In cells, however, Dbh does not appear to contribute significantly to spontaneous frameshifts in these same sequence contexts. This suggests that either the error-prone DNA synthesis activity of Dbh is reduced in vivo and/or Dbh is restricted from replicating these sequences. Here, we test the hypothesis that the propensity for Dbh to make single base deletion mutations is reduced through interaction with the S. acidocaldarius heterotrimeric sliding clamp processivity factor, PCNA-123. We first confirm that Dbh physically interacts with PCNA-123, with the interaction requiring both the PCNA-1 subunit and the C-terminal 10 amino acids of Dbh, which contain a predicted PCNA-interaction peptide (PIP) motif. This interaction stimulates the polymerase activity of Dbh, even on short, linear primer-template DNA by increasing the rate of nucleotide incorporation. This stimulation requires an intact PCNA-123 heterotrimer and a DNA duplex length of at least 18 basepairs, the minimal length predicted from structural data to bind to both the polymerase and the clamp. Finally, we find that PCNA-123 increases the fidelity of Dbh on a single-base deletion hotspot sequence 3-fold by promoting an increase in the rate of correct, but not incorrect, nucleotide addition and propose that PCNA-123 induces Dbh to adopt a more active conformation that is less prone to creating deletions during DNA synthesis. HighlightsO_LIPCNA increases the fidelity of Dbh polymerase on a deletion-hotspot sequence. C_LIO_LIThe interaction stimulates incorporation of the correct, but not incorrect, nucleotide. C_LIO_LIA minimal duplex length of 18 bp is required for PCNA to stimulate polymerase activity. C_LIO_LIStructural modeling suggests that PCNA induces a conformational change in Dbh. C_LI

biochemistry↗