Biology subjects

Brown, A.

Publications and source records attributed to Brown, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

The glucocorticoid receptor is a critical regulator of muscle satellite cell quiescence.

Glucocorticoids are powerful anti-inflammatory medications that are associated with muscle atrophy. The effect of glucocorticoids in myofibers is well-studied, yet the role of the glucocorticoid receptor (GR), the primary mediator of glucocorticoid transcriptional responses, and the impact of glucocorticoid signalling in muscle stem cells (MuSCs), the adult progenitors responsible for regeneration, remain unknown. We developed a conditional null mouse model to knock out glucocorticoid receptor (GR) expression in MuSCs (GRMuSC-/-) and established that while GR is dispensable for muscle regeneration, it is a critical regulator of MuSC homeostasis. Loss of GR significantly increased cycling MuSCs as compared to controls in injury-naive mice and on single EDL myofiber cultures, and as such, loss of GR in MuSCs leads to precocious activation and subsequent proliferation as compared to controls. Bulk RNA-sequencing from in situ fixed MuSCs from injury-naive GRMuSC-/- muscle identified a gene signature consistent with cells that have exited quiescence and undergone activation, with evidence of sexual dimorphism. Using ATAC-seq and footprinting we identify putative GR targets that promote quiescence. Thus, we advance the GR as a previously unrecognized crucial transcriptional regulator of gene expression in MuSCs whose activity is highest in quiescent cells and is essential to maintain that state.

cell biology↗

bioRxiv2023

An allelic series at the EDNRB2 locus controls diverse piebalding patterns in the domestic pigeon

Variation in pigment patterns within and among vertebrate species reflects underlying changes in cell migration and function that can impact health, reproductive success, and survival. The domestic pigeon (Columba livia) is an exceptional model for understanding the genetic changes that give rise to diverse pigment patterns, as selective breeding has given rise to hundreds of breeds with extensive variation in plumage color and pattern. Here, we map the genetic architecture of a suite of pigmentation phenotypes known as piebalding. Piebalding is characterized by patches of pigmented and non-pigmented feathers, and these plumage patterns are often breed-specific and stable across generations. Using a combination of quantitative trait locus mapping in F2 laboratory crosses and genome-wide association analysis, we identify a locus associated with piebalding across many pigeon breeds. This shared locus harbors a candidate gene, EDNRB2, that is a known regulator of pigment cell migration, proliferation, and survival. We discover multiple distinct haplotypes at the EDNRB2 locus in piebald pigeons, which include a mix of protein-coding, noncoding, and structural variants that are associated with depigmentation in specific plumage regions. These results identify a role for EDNRB2 in pigment patterning in the domestic pigeon, and highlight how repeated selection at a single locus can generate a diverse array of stable and heritable pigment patterns. AUTHOR SUMMARYBoth wild and domestic birds show striking variation in pigment patterning, and these pigment patterns can play critical roles in mate choice, communication, and camouflage. Despite the importance of pigment patterning for survival and reproductive success, the mechanisms that control pigment patterning remain incompletely understood. In domestic pigeons, artificial selection has given rise to a wide array of pigmentation patterns within a single species, including a suite of phenotypes called "piebalding" that is characterized by regional loss of feather pigment. Here, we took advantage of the wide array of distinct piebalding phenotypes in domestic pigeons to map the genetic basis of region-specific loss of plumage pigment. Rather than focusing on a single piebalding pattern, we sought to broadly understand genetic control of regional pigment loss by examining several breeds with different piebalding patterns. We compared the genomes of piebald and non-piebald pigeons using both genetic crosses and genome-wide association studies, and identified several genetic changes affecting the endothelin receptor gene EDNRB2. Our findings highlight how independent mutations at a single locus can drive diversification of plumage pigment patterning within a species.

genetics↗

bioRxiv2023

Efficacy of the oral nucleoside prodrug GS-5245 (Obeldesivir) against SARS-CoV-2 and coronaviruses with pandemic potential

Despite the wide availability of several safe and effective vaccines that can prevent severe COVID-19 disease, the emergence of SARS-CoV-2 variants of concern (VOC) that can partially evade vaccine immunity remains a global health concern. In addition, the emergence of highly mutated and neutralization-resistant SARS-CoV-2 VOCs such as BA.1 and BA.5 that can partially or fully evade (1) many therapeutic monoclonal antibodies in clinical use underlines the need for additional effective treatment strategies. Here, we characterize the antiviral activity of GS-5245, Obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved RNA-dependent viral RNA polymerase (RdRp). Importantly, we show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-related Bat-CoV RsSHC014, Middle East Respiratory Syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant in vitro and highly effective as antiviral therapy in mouse models of SARS-CoV, SARS-CoV-2 (WA/1), MERS-CoV and Bat-CoV RsSHC014 pathogenesis. In all these models of divergent coronaviruses, we observed protection and/or significant reduction of disease metrics such as weight loss, lung viral replication, acute lung injury, and degradation in pulmonary function in GS-5245-treated mice compared to vehicle controls. Finally, we demonstrate that GS-5245 in combination with the main protease (Mpro) inhibitor nirmatrelvir had increased efficacy in vivo against SARS-CoV-2 compared to each single agent. Altogether, our data supports the continuing clinical evaluation of GS-5245 in humans infected with COVID-19, including as part of a combination antiviral therapy, especially in populations with the most urgent need for more efficacious and durable interventions.

microbiology↗

bioRxiv2023

Present: flexible neuroscience- and technology-driven frameworks for the study of classroom engagement

Classroom engagements impact on academic success is crucial. However, the contributions of affective, cognitive, and behavioral components of engagement remain uncertain. We conducted two studies using non-invasive, research-based approaches to clarify these contributions. Study 1 employed portable EEG headsets to measure cognitive engagement, in-class quizzes assessed content retention, and post-class subjective questionnaires indexed affective engagement by measuring feelings of learning and engagement. Content retention predicted subjective measures, while the EEG theta/beta ratio was negatively related to content retention but positively related to subjective measures. Study 2 featured embedded measures of content retention, confidence, engagement, background knowledge, and indexed behavioral engagement looking at nonverbal behavior quantified via video camera recordings. Confidence and engagement were significantly correlated with each other and with particular facial muscle, gaze direction, and head pose movements. We discuss how these approaches enable real-time studies of classroom engagement and can be integrated to develop neurofeedback interventions.

neuroscience↗

bioRxiv2023

Identification of proteins that bind extracellular microRNAs secreted by the parasitic nematode Trichinella spiralis

Small non-coding RNAs such as microRNAs (miRNAs) are conserved across eukaryotes and play key roles in regulating gene expression. In many organisms, miRNAs are also secreted from cells, often encased within vesicles such as exosomes and sometimes extravesicular. The mechanisms of miRNA secretion, how they are stabilised outside of cells and their functional importance are poorly understood. Recently we characterised the parasitic nematode Trichinella spiralis as a model to study miRNA secretion. T. spiralis larvae secrete abundant miRNAs which are largely extravesicular. Here, we investigated how T. spiralis miRNAs might remain stable outside of cells. Using proteomics, we identified two RNA binding proteins secreted by T. spiralis larvae and characterised their RNA binding properties. One, a homologue of the known RNA binding protein KSRP, binds miRNA in a selective and sequence-specific fashion. Another protein, which is likely a novel RNA binding protein, binds non-selectively to miRNA. Our results suggest a possible mechanism for miRNA secretion by T. spiralis and may have relevance for understanding the biology of extracellular miRNA more widely.

biochemistry↗

bioRxiv2023

Vaccine-mediated protection against merbecovirus and sarbecovirus challenge in mice

The emergence of three distinct highly pathogenic human coronaviruses - SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019 - underlines the need to develop broadly active vaccines against the Merbecovirus and Sarbecovirus betacoronavirus subgenera. While SARS-CoV-2 vaccines are highly protective against severe COVID-19 disease, they do not protect against other sarbecoviruses or merbecoviruses. Here, we vaccinate mice with a trivalent sortase-conjugate nanoparticle (scNP) vaccine containing the SARS-CoV-2, RsSHC014, and MERS-CoV receptor binding domains (RBDs), which elicited live-virus neutralizing antibody responses and broad protection. Specifically, a monovalent SARS-CoV-2 RBD scNP vaccine only protected against sarbecovirus challenge, whereas the trivalent RBD scNP vaccine protected against both merbecovirus and sarbecovirus challenge in highly pathogenic and lethal mouse models. Moreover, the trivalent RBD scNP elicited serum neutralizing antibodies against SARS-CoV, MERS-CoV and SARS-CoV-2 BA.1 live viruses. Our findings show that a trivalent RBD nanoparticle vaccine displaying merbecovirus and sarbecovirus immunogens elicits immunity that broadly protects mice against disease. This study demonstrates proof-of-concept for a single pan-betacoronavirus vaccine to protect against three highly pathogenic human coronaviruses spanning two betacoronavirus subgenera.

immunology↗

bioRxiv2023

Automated model building and protein identification in cryo-EM maps

Interpreting electron cryo-microscopy (cryo-EM) maps with atomic models requires high levels of expertise and labour-intensive manual intervention. We present ModelAngelo, a machine-learning approach for automated atomic model building in cryo-EM maps. By combining information from the cryo-EM map with information from protein sequence and structure in a single graph neural network, ModelAngelo builds atomic models for proteins that are of similar quality as those generated by human experts. For nucleotides, ModelAngelo builds backbones with similar accuracy as humans. By using its predicted amino acid probabilities for each residue in hidden Markov model sequence searches, ModelAngelo outperforms human experts in the identification of proteins with unknown sequences. ModelAngelo will thus remove bottlenecks and increase objectivity in cryo-EM structure determination.

molecular biology↗

bioRxiv2023

ADSC-EVs modulate primary human macrophages to an anti-inflammatory phenotype in vitro

BackgroundEVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue repair and regeneration because of their purported immune-regulatory properties. In this capacity, ADSC-EVs could be beneficial in improving graft retention rates for autologous fat grafting (AFG) post-mastectomy as, currently, grafted tissue rates are reported to be variable and low. Enriching grafted tissue with ADSC-EVs may improve retention rates by modulating macrophages resident within both the breast and lipoaspirate. We aimed to identify key macrophage phenotypes that are modulated by ADSC-EVs in vitro. MethodsADSCs were isolated from lipoaspirates of women undergoing AFG and characterised by flow cytometry and differentiation potential. ADSC-EVs were isolated from cell culture media and characterised by tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and Western blot. Primary monocyte-derived macrophages were polarized to an M1-like (GM-CSF, IFN{gamma}) or M2-like phenotype (M-CSF, IL-4) or maintained (M0-like; M-CSF) and, at the time of polarization, ADSC-EVs were co-cultured with macrophages for 48 hrs. Flow cytometry coupled with high-dimensional analysis was used to cluster macrophages post co-culture. A manual gating strategy was generated to recapitulate these clusters and was applied to a repeat experimental run. Both runs were analysed to examine the prevalence of each cluster, representing a unique macrophage phenotype, with and without ADSC-EVs. ResultsFollowing the addition of ADSC-EVs, M0-like macrophages demonstrated a reciprocal shift of cell distribution from a cluster defined as having a high inflammatory profile (CD36+++CD206+++CD86+++; 38.6{+/-}14.8% of M1-like macrophages without ADSC-EVs; 16.5{+/-}7.0% with ADSC-EVs; p<0.0001) to a cluster with a lower inflammatory profile (CD36+CD206+CD86+; 16.6{+/-}11.2% to 35{+/-}21.5%; p<0.05). There was no shift in M2-like clusters following treatment with ADSC-EVs. ConclusionsADSC-EVs are complex regulators of macrophage phenotype that can shift macrophages away from a heightened pro-inflammatory state.

molecular biology↗

bioRxiv2023

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets. HighlightsO_LIRibo-seq reveals widespread translation of non-canonical ORFs in medulloblastoma C_LIO_LIHigh-resolution CRISPR tiling reveals uORF functions in medulloblastoma C_LIO_LIASNSD1-uORF controls downstream pathways with the prefoldin-like complex C_LIO_LIASNSD1-uORF is necessary for medulloblastoma cell survival C_LI

cancer biology↗

bioRxiv2023

Synthesis and validation of click-modified of NOD1/2 agonists

NOD1 and NOD2 sense small bacterial peptidoglycan fragments often called muropeptides. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The clickchemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.

immunology↗

bioRxiv2023

Vascular Smooth Muscle Cell Mechanotransduction Through Serum and Glucocorticoid Inducible Kinase-1 Promotes Interleukin-6 Production and Macrophage Accumulation in Murine Hypertension

ObjectiveThe objective of this investigation was to demonstrate that in vivo induction of hypertension (HTN) and in vitro cyclic stretch of aortic VSMCs can cause SGK-1-dependent production of cytokines to promote macrophage accumulation as agents of vascular remodeling. MethodsHTN was induced in C57Bl/6 mice with AngiotensinII (AngII) infusion (1.46mg/kg/day x 21 days) with or without systemic infusion of EMD638683 (2.5mg/kg/day x 21 days), a selective SGK-1 inhibitor. Systolic blood pressure (SBP) was recorded on days 0 and 21. At terminal study, abdominal aortas were harvested to quantify SGK-1 activity (pSGK-1:SGK-1) by immunoblot. Additional replicates were digested and analyzed by flow cytometry for abundance of CD11b+/F4-80+ cells (macrophages). Plasma was analyzed by ELISA to quantify IL-6 and MCP-1. Aortic VSMCs from wild-type (WT) mice were subjected to 12% biaxial cyclic stretch for 3 or 12 hours +/- EMD638683 (10M) and +/- SGK-1siRNA with subsequent QPCR for IL-6 and MCP-1 expression. Culture media was analyzed by ELISA for IL-6 and MCP-1. Aortic VSMCs from SGK-1flox+/+ mice were transfected with Cre-Adenovirus to knockout SGK-1 (SGK-1KO VSMCs) and underwent parallel tension experimentation. Computational modeling was employed to simulate VSMC signaling due to mechanical strain and AngII. Statistical analysis included ANOVA with significance at p<0.05. ResultsSGK-1 activity (pSGK-1:SGK-1) was upregulated in the abdominal aorta of mice with HTN and significantly reduced by treatment with EMD638683. Concurrently, increased CD11b+/F4-80+ cells and plasma IL-6 levels in the HTN group and reduction with EMD638683 was observed. This mirrored the increased abundance of IL-6 in media from Stretch WT VSMCs, and attenuation of the effect with EMD638683. Treating WT VSMCs with SGK-1siRNA likewise inhibited IL-6 expression. MCP-1 also demonstrated increased expression and secretion into the media in WT VSMCs with Stretch. Further supporting the integral role of mechanical signaling through SGK-1, target gene expression and cytokine secretion was unchanged in SGK-1KO VSMCs with Stretch, and computer modeling confirmed SGK-1 as an intersecting node of signaling due to mechanical strain and AngII. In summation, this data suggests a biomechanical link between aortic VSMC mechanotransduction and cytokine production to promote macrophage accumulation, mediated in-part by SGK-1 activation. ConclusionMechanotransduction through SGK-1 is instrumental in pro-inflammatory cytokine production and aortic macrophage accumulation in systemic HTN, therefore further investigation into targeting this kinase may present opportunities to modulate hypertensive vascular remodeling.

physiology↗

bioRxiv2023

Spatiotemporal Dynamics of Vcam1 Regulates Cholangiocarcinoma Mass Expansion and Tumor Dissemination under Growth-suppressive Peritumoral Myofibroblasts

Intrahepatic cholangiocarcinoma (iCCA) is characterized by its highly desmoplastic stroma. Myofibroblasts (MFs) are present both within the tumor mass (intratumoral MFs, iMFs) and at the tumor border (peritumoral MFs, pMFs). Using a spheroid-based coculture system, we show that the initial iCCA-pMF contact is growth suppressive to the tumor cells. However, prolonged iCCA-pMF interaction elicits significant tumor cell invasion and dissemination. We find that vascular cell adhesion molecule-1 (Vcam1) level is elevated in tumor cells in contact with pMFs but low in disseminated tumor cells both in vitro and in vivo. A gene regulatory network analysis of mouse and patient iCCA tumors and Vcam1 knockout (Vcam1KO) demonstrate a heavy involvement of Vcam1 in epithelial-to-mesenchymal transition. While Vcam1KO has only a limited impact on tumor cell growth in their monoculture, Vcam1KO spheroids exhibit instant dissemination and a severe growth defect when cocultured with pMFs. When transplanted into the liver, Vcam1KO iCCA cells show a similar increase in dissemination but a significant defect in establishing primary and metastatic tumors. Incomplete blocking of Vcam1 in vivo reduces the size but increase the number of metastatic lesions. Overall, our study shows a spatiotemporal regulation of iCCA growth and dissemination by pMFs in a Vcam1-dependent manner.

cancer biology↗

bioRxiv2023

Ribonucleotide Reductase Subunit Switching in Hepatoblastoma Drug Response and Relapse

Prognosis of children with high-risk hepatoblastoma (HB), the most common pediatric liver cancer, remains poor. In this study, we found ribonucleotide reductase (RNR) subunit M2 (RRM2) was one of the key genes supporting cell proliferation in high-risk HB. While standard chemotherapies could effectively suppress RRM2 in HB cells, they induced a significant upregulation of the other RNR M2 subunit, RRM2B. Computational analysis revealed distinct signaling networks RRM2 and RRM2B were involved in HB patient tumors, with RRM2 supporting cell proliferation and RRM2B participating heavily in stress response pathways. Indeed, RRM2B upregulation in chemotherapy-treated HB cells promoted cell survival and subsequent relapse, during which RRM2B was gradually replaced back by RRM2. Combining an RRM2 inhibitor with chemotherapy showed an effective delaying of HB tumor relapse in vivo. Overall, our study revealed the distinct roles of the two RNR M2 subunits and their dynamic switching during HB cell proliferation and stress response.

cancer biology↗

bioRxiv2023

Mutual antagonism between glucocorticoid and canonical Wnt signaling pathways in B-cell acute lymphoblastic leukemia

Glucocorticoids (GCs; i.e., steroids) are important chemotherapeutic agents in the treatment of B-cell precursor acute lymphoblastic leukemia (B-ALL) and de novo GC resistance predicts relapse and poor clinical outcome in patients. Glucocorticoids induce B-ALL cell apoptosis through activation of glucocorticoid receptor (GR), a ligand-induced nuclear receptor transcription factor (TF). We previously identified disruptions to glucocorticoid receptor (GR)-bound cis-regulatory elements controlling TLE1 expression in GC-resistant primary B-ALL cells from patients. TLE1 is a GC-response gene up-regulated by steroids and functions as a canonical Wnt signaling repressor. To better understand the mechanistic relationship between GC signaling and canonical Wnt signaling, we performed diverse functional analyses that identified extensive crosstalk and mutual antagonism between these two signaling pathways in B-ALL. We determined that crosstalk and antagonism was driven by the binding of GR and the canonical Wnt signaling TFs LEF1 and TCF7L2 to overlapping sets of cis-regulatory elements associated with genes impacting cell death and cell proliferation, and was further accompanied by overlapping and opposing transcriptional programs. Our data additionally suggest that cis-regulatory disruptions at TLE1 are linked to GC resistance through a dampening of the GC response and GC-mediated apoptosis via enhanced canonical Wnt signaling. As a result of the extensive genomic and gene regulatory connectivity between these two signaling pathways, our data supports the importance of canonical Wnt signaling in mediating GC resistance in B-ALL.

cancer biology↗

bioRxiv2023

Unlocking de novo antibody design with generative artificial intelligence

Generative AI has the potential to redefine the process of therapeutic antibody discovery. In this report, we describe and validate deep generative models for the de novo design of antibodies against human epidermal growth factor receptor (HER2) without additional optimization. The models enabled an efficient workflow that combined in silico design methods with high-throughput experimental techniques to rapidly identify binders from a library of [~]106 heavy chain complementarity-determining region (HCDR) variants. We demonstrated that the workflow achieves binding rates of 10.6% for HCDR3 and 1.8% for HCDR123 designs and is statistically superior to baselines. We further characterized 421 diverse binders using surface plasmon resonance (SPR), finding 71 with low nanomolar affinity similar to the therapeutic anti-HER2 antibody trastuzumab. A selected subset of 11 diverse high-affinity binders were functionally equivalent or superior to trastuzumab, with most demonstrating suitable developability features. We designed one binder with [~]3x higher cell-based potency compared to trastuzumab and another with improved cross-species reactivity1. Our generative AI approach unlocks an accelerated path to designing therapeutic antibodies against diverse targets.

synthetic biology↗

bioRxiv2022

30x30 biodiversity gains rely on national coordination

Protecting 30% of land by 2030 is an invaluable opportunity to combat the ongoing biodiversity crisis, but critical questions remain regarding what biodiversity to prioritize, how to coordinate protection, and how to incorporate global change. Here, we evaluate how well different 30x30 expansion scenarios capture the climatically viable ranges of Canadian terrestrial vertebrates, plants, and butterflies. We find that national coordination protects vastly more biodiversity (65% of species; 40% of species-at-risk) than regional approaches, which safeguard at least 33% fewer species and 75% fewer species-at-risk. Whereas prioritizing different taxa or biodiversity facets (e.g., phylogenetic diversity) incur smaller trade-offs. Surprisingly, national priorities closely match transnational ones, indicating that national coordination could efficiently contribute to global targets while protecting Canadas biodiversity at large.

ecology↗

bioRxiv2022

High Density Domain-Focused CRISPR Screens Reveal Epigenetic Regulators of HOX/MEIS Gene Expression in Acute Myeloid Leukemia

Aberrant expression of stem-cell-associated genes is a common feature in acute myeloid leukemia (AML) and is linked to leukemic self-renewal and therapy resistance. Using AF10-rearranged leukemia as a prototypical example displaying a recurrent "stemness" network activated in AML, we screened for chromatin regulators that sustain aberrant activation of these networks. We deployed a CRISPR-Cas9 screen with a bespoke domain-focused library and identified several novel chromatin-modifying complexes as regulators of the TALE domain transcription factor MEIS1, a key leukemia stem cell (LSC)-associated gene. CRISPR droplet sequencing revealed that many of these MEIS1 regulators coordinately controlled the transcription of several AML oncogenes. In particular, we identified a novel role for the Tudor-domain containing chromatin reader protein SGF29 in the transcription of key AML oncogenes. Furthermore, SGF29 deletion impaired leukemogenesis in models representative of multiple AML subtypes. Our studies reveal a novel role for SGF29 as a non-oncogenic dependency in AML and identify the SGF29 Tudor domain as an attractive target for drug discovery.

cancer biology↗

bioRxiv2022

Germline variation contributes to false negatives in CRISPR-based experiments with varying burden across ancestries

Reducing disparities is critical to promote equity of access to precision treatments for all patients with cancer. While socioenvironmental factors are a major driver behind such disparities, biological differences also are likely to contribute. The prioritization of cancer drug targets is foundational for drug discovery, yet whether ancestry-related signals in target discovery pipelines exist has not been systematically explored due to the absence of data at the appropriate scale. Here, we analyzed data from 611 genome-scale CRISPR/Cas9 viability experiments in human cell line models as part of the Cancer Dependency Map to identify ancestry-associated genetic dependencies. Surprisingly, we found that most putative associations between ancestry and dependency arose from artifacts related to germline variants that are present at different frequencies across ancestry groups. In 2-5% of genes profiled in each cellular model, germline variants in sgRNA targeting sequences likely reduced cutting by the CRISPR/Cas9 nuclease. Unfortunately, this bias disproportionately affected cell models derived from individuals of recent African descent because their genomes tended to diverge more from the consensus genome typically used for CRISPR/Cas9 guide design. To help the scientific community begin to resolve this source of bias, we report three complementary methods for ancestry-agnostic CRISPR experiments. This report adds to a growing body of literature describing ways in which ancestry bias impacts cancer research in underappreciated ways.

cancer biology↗