Genome-wide Screening Approach Research Articles

Genome-wide CRISPR screening identifies the pivotal role of ANKRD42 in colorectal cancer metastasis through EMT regulation.

Colorectal cancer (CRC), a pervasive and lethal malignancy of gastrointestinal cancer, imposes significant challenges due to the occurrence of distant metastasis in advanced stages. Understanding the intricate regulatory mechanisms driving CRC distant metastasis is of paramount importance. CRISPR-Cas9 screening has emerged as a powerful tool for investigating tumor initiation and progression. However, its application in studying CRC distant metastasis remains largely unexplored. To establish a model that faithfully recapitulates CRC liver metastasis in patients, we developed an in vivo genome-wide CRISPR-Cas9 screening approach using a spleen-injected liver metastasis mouse model. Through comprehensive screening of a whole-genome sgRNA library, we identified ANKRD42 as a pivotal regulatory gene facilitating CRC liver metastasis. Analysis of the TCGA database and our clinical cohorts unveiled heightened ANKRD42 expression in metastases. At the cellular level, the attenuation of ANKRD42 impaired the migration and invasion processes of tumor cells. In vivo experiments further validated these observations, highlighting the diminished liver metastatic capacity of tumor cells upon ANKRD42 knockdown. To unravel the specific mechanisms by which ANKRD42 regulates CRC distant metastasis, we leveraged patient-derived organoid (PDO) models. Depleting ANKRD42 in PDOs sourced from liver metastases precipitated the downregulation of pivotal genes linked to epithelial-mesenchymal transition (EMT), including CDH2 and SNAI2, thereby effectively suppressing tumor metastasis. This study not only establishes a conceptual framework but also identifies potential therapeutic avenues for advanced-stage distant metastasis in CRC patients.

Genome-Wide Screening Approaches for Biochemical Reactions Independent of Cell Growth.

Genome-wide screening is a potent approach for comprehensively understanding the molecular mechanisms of biological phenomena. However, despite its widespread use in the past decades across various biological targets, its application to biochemical reactions with temporal and reversible biological outputs remains a formidable challenge. To uncover the molecular machinery underlying various biochemical reactions, we have recently developed the revival screening method, which combines flow cytometry-based cell sorting with library reconstruction from collected cells. Our refinements to the traditional genome-wide screening technique have proven successful in revealing the molecular machinery of biochemical reactions of interest. In this article, we elucidate the technical basis of revival screening, focusing on its application to CRISPR-Cas9 single guide RNA (sgRNA) library and complementary DNA (cDNA) library screening. Finally, we also discuss the future of genome-wide screening while describing recent achievements from in vitro and in vivo screening.

Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks.

Bacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus. We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (∆fimE) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.IMPORTANCEType 6 secretion systems (T6SS) are molecular weapons employed by gram-negative bacteria to eliminate neighboring microbes. T6SS plays a pivotal role as a virulence factor, enabling pathogenic gram-negative bacteria to compete with the established communities to colonize hosts and induce infections. Gaining a deeper understanding of bacterial interactions will allow the development of strategies to control the action of systems such as the T6SS that can manipulate bacterial communities. In this context, we demonstrate that bacteria targeted by T6SS attacks from the enteric pathogen Cronobacter malonaticus, which poses a significant threat to infants, can develop a collective protective mechanism centered on the production of type I fimbriae. These adhesive structures promote the aggregation of bacterial preys and the formation of microcolonies, which protect the cells from T6SS attacks.

PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a common cause of familial ALS, accounting for around 4% of the cases. Understanding the mechanisms by which mutant FUS becomes toxic to neurons can provide insight into the pathogenesis of both familial and sporadic ALS. We have previously observed that overexpression of wild-type or ALS-mutant FUS in Drosophila motor neurons is toxic, which allowed us to screen for novel genetic modifiers of the disease. Using a genome-wide screening approach, we identified Protein Phosphatase 2A (PP2A) and Glycogen Synthase Kinase 3 (GSK3) as novel modifiers of FUS-ALS. Loss of function or pharmacological inhibition of either protein rescued FUS-associated lethality in Drosophila. Consistent with a conserved role in disease pathogenesis, pharmacological inhibition of both proteins rescued disease-relevant phenotypes, including mitochondrial trafficking defects and neuromuscular junction failure, in patient iPSC-derived spinal motor neurons (iPSC-sMNs). In FUS-ALS flies, mice, and human iPSC-sMNs, we observed reduced GSK3 inhibitory phosphorylation, suggesting that FUS dysfunction results in GSK3 hyperactivity. Furthermore, we found that PP2A acts upstream of GSK3, affecting its inhibitory phosphorylation. GSK3 has previously been linked to kinesin-1 hyperphosphorylation. We observed this in both flies and iPSC-sMNs, and we rescued this hyperphosphorylation by inhibiting GSK3 or PP2A. Moreover, increasing the level of kinesin-1 expression in our Drosophila model strongly rescued toxicity, confirming the relevance of kinesin-1 hyperphosphorylation. Our data provide in vivo evidence that PP2A and GSK3 are disease modifiers, and reveal an unexplored mechanistic link between PP2A, GSK3, and kinesin-1, that may be central to the pathogenesis of FUS-ALS and sporadic forms of the disease.

Abstract A043: Identifying critical vulnerabilities that sensitise Lkb1-mutant lung adenocarcinoma to T cell mediated killing

Abstract KRAS is the most frequently altered oncogene in lung adenocarcinoma (LUAD). While the clinical development of targeted therapies has improved the treatment of a subset of patients, the heterogeneity of KRAS mutant LUAD remains a clinical challenge. Identifying tumor intrinsic dependencies through common co-occurring mutations are one approach to more effectively target KRAS-mutant LUAD. Loss-of-function mutations in STK11/Lkb1, are frequently found co-mutated with KRAS, and comprise an aggressive form of the disease. Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis, which aims to unleash CD8 effector T cells against tumors, are currently used as a first-line treatment strategy for KRAS mutant patients. However, KRAS mutant tumors that harbour co-mutations in STK11 fail to respond to ICIs. A greater understanding of the tumor-intrinsic processes that allow STK11/Lkb1 mutant tumors to evade immune detection is required to enhance the response of ICIs and improve patient outcomes. Here, we have used an unbiased genome-wide CRISPR knockout screening approach in lung cancer cell lines generated from the Kras G12D/+/Lkb1 fl/fl (KL) mouse model to unveil tumor-intrinsic mechanisms involved in anti-tumor immunity. To identify gene targets that sensitise tumor cells to T cell mediated killing, a co-culture method using the OVA/OT-I system was employed. Importantly, sgRNAs targeting genes encoding components of interferon signalling (Jak1, Stat1, Ifngr1) and MHC-I presentation (B2m, H2-k1) were enriched following serial application of OT-I T cells, consistent with the role these pathways play in resistance to ICIs. Conversely, sgRNAs targeting several genes were found to sensitise KL tumor cells to T cell mediated killing. This included genes (Serpinb9, Ptpn2) previously implicated in increasing T cell-mediated tumor cell killing, supporting the robustness of our screening platform. Excitingly, sgRNAs targeting novel genes, not previously implicated in mediating anti-tumor immunity were also identified. Current investigations validating the ability of candidate genes to sensitise KL tumor cells to T cell killing will be presented. Together, these results demonstrate the power of whole genome CRISPR screens in identifying candidate genes that may serve as therapeutic targets to improve treatment responses in KRAS/STK11/Lkb1 mutant LUAD patients. Citation Format: Jackson A McDonald, Danielle Boyd, Sarah Diepstraten, Leanne Scott, Lin Tai, Andrew Kueh, Marian Burr, Sarah A Best, Marco J Herold, Kate D Sutherland. Identifying critical vulnerabilities that sensitise Lkb1-mutant lung adenocarcinoma to T cell mediated killing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A043.

Combined in vitro/in vivo genome-wide CRISPR screens in triple negative breast cancer identify cancer stemness regulators in paclitaxel resistance

Triple negative breast cancer (TNBC) is defined as lacking the expressions of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC patients exhibit relatively poor clinical outcomes due to lack of molecular markers for targeted therapies. As such chemotherapy often remains the only systemic treatment option for these patients. While chemotherapy can initially help shrink TNBC tumor size, patients eventually develop resistance to drug, leading to tumor recurrence. We report a combined in vitro/in vivo genome-wide CRISPR synthetic lethality screening approach in a relevant TNBC cell line model to identify several targets responsible for the chemotherapy drug, paclitaxel resistance. Computational analysis integrating in vitro and in vivo data identified a set of genes, for which specific loss-of-function deletion enhanced paclitaxel resistance in TNBC. We found that several of these genes (ATP8B3, FOXR2, FRG2, HIST1H4A) act as cancer stemness negative regulators. Finally, using in vivo orthotopic transplantation TNBC models we showed that FRG2 gene deletion reduced paclitaxel efficacy and promoted tumor metastasis, while increasing FRG2 expression by means of CRISPR activation efficiently sensitized TNBC tumors to paclitaxel treatment and inhibited their metastatic abilities. In summary, the combined in vitro/in vivo genome-wide CRISPR screening approach proved effective as a tool to identify novel regulators of paclitaxel resistance/sensitivity and highlight the FRG2 gene as a potential therapeutical target overcoming paclitaxel resistance in TNBC.

Incorporating long non-coding RNA (lncRNA) into genome-wide biomarker screening for prognostic gene signatures of immunotherapy outcomes.

2617 Background: Emerging data have shown that long non-coding RNA (lncRNAs) have the potential to become biomarkers in cancers with potential roles in regulating cell proliferation, apoptosis, migration, invasion and maintenance of stemness during cancer development. In this research, we aimed for a genome-wide biomarker screening approach incorporating lncRNA in order to identify key molecules and pathways involved in ICI response and resistance. Methods: We utilized clinical and transcriptomic data collected under the Total Cancer Care Protocol (NCT03977402) and Avatar project within the Oncology Research Information Exchange Network (ORIEN). We analyzed RNA-seq data from a total of 875 ICI treated pts. A novel resampling-based technique with elastic net regression was utilized for genome-wide biomarker screening, in which Cox regression was used for survival outcomes and logistic regression for a 2-year-based dichotomized outcome. All search was done through 200 resampling steps, each consisting of randomly selecting 80% of pts and 80% of all gene signatures. Moreover, we searched the genes that are most complementary to the established immune cell scores (i.e., Ecotyper and xCell) by incorporating these scores into the regression adjustment. Finally, we applied the same approach to identify prognostic long non-coding RNA (lncRNA) signatures. All analyses were carried out on all ICI pts, as well as on subgroups stratified by cancer type. Results: Of 875 pts treated with ICI, 125 had melanoma, 143 had kidney cancer, 126 had non-small lung cancer, and 110 had head and neck cancer. The genome-wide selection method successfully identified a panel of top prognostic genes and pathways for all cancer types included. In melanoma, the top five prognostic genes were ZDHHC2, CBX4, MET, NTN4, and HLA-DQA1, with 80%, 78%, 77.5%, 77.5%, 76.5% resampling-based selection probability respectively; the top five lncRNAs were AC073655.3, AC116348.3, PITPNA-AS1, AC011447.3, and AL021978.1. When adjusting for the immune scores in the selection model, the top five prognostic protein-coding genes in melanoma were TENM3, POLR2J3, SCCPDH, MET and SIAH1, and the top five lncRNAs were AC007620.2, PITPNA-AS1, HCG11, SLC25A5-AS1, and LINC02258, respectively. Conclusions: Our analysis has successfully identified gene panels of mRNA and lncRNA molecules with potential roles related to ICI mechanisms of action. Ongoing analyses are further investigating the potential mechanisms and pathways linking these molecules and will be presented at the meeting. [Table: see text]

Ubiquitin-Mediated Regulation of Autophagy During Viral Infection

Virus infections skew the host autophagic response to meet their replication and transmission demands by tapping into the critical host regulatory mechanisms that control the autophagic flux. This review is a compendium of previous reports highlighting the mechanisms that viruses adapt to hijack the host ubiquitination machinery to repurpose autophagy for their sustenance. Emerging evidence suggests a critical role of host ubiquitin machinery in the manifestation of the antiviral or proviral functions of autophagy. Lately, more emphasis has been laid to identify specific host E3 ubiquitin ligases, their targets (viral or host), and characterizing corresponding ubiquitin linkages by biochemical or genome-wide genetic screening approaches. Here, we highlight how viruses ingeniously engage and subvert the host ubiquitin-autophagy system to promote virus replication and antagonize intracellular innate immune responses.

CRISPR Activation/Interference Screen to Identify Genetic Networks in HDAC-Inhibitor-Resistant Cells.

Epigenetic alterations have been identified in various tumor types. In part, these alterations are mediated via increased histone deacetylase activity. Although preclinical results of monotherapies with histone deacetylase inhibitors (HDACi) are promising, success in clinical trials is limited. Reasons for these limitations may be de novo or acquired resistance to HDAC inhibitors that could be overcome with rational combination therapies. This requires knowledge of resistance mechanism along with the involved genetic networks. One way to identify such genetic networks is the implementation of a CRISPR-based technology allowing transcriptional repression (CRISPRi) and activation (CRISPRa) at a genome-wide scale. We describe a simple approach to amplify and validate sgRNA libraries, generate a myeloid progenitor cell line expressing catalytically dead Cas9 (dCas9) fusion proteins with transcriptional effectors to repress or activate genetic regions of interest and demonstrate a complementary genome-wide HDACi resistance screening approach. Furthermore, we present bioinformatics tools for quality control and analysis of the sequencing data.

CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor.

Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.

NUDT21 limits CD19 levels through alternative mRNA polyadenylation in B cell acute lymphoblastic leukemia.

B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.

Assessment of genotoxic chemicals using chemogenomic profiling based on gene-knockout library in Saccharomyces cerevisiae

Understanding the adverse effects of genotoxic chemicals and identifying them effectively from non-genotoxic chemicals are of great worldwide concerns. Here, Saccharomyces cerevisiae (yeast) genome-wide single-gene knockout screening approach was conducted to assess two genotoxic chemicals (4-nitroquinoline-1-oxide (4-NQO) and formaldehyde (FA)) and environmental pollutant dichloroacetic acid (DCA, genotoxicity is controversial). DNA repair was significant enriched in the gene ontology (GO) biology process (BP) terms and KEGG pathways when exposed to low concentrations of 4-NQO and FA. Higher concentrations of 4-NQO and FA influenced some RNA metabolic and biosynthesis pathways. Moreover, replication and repair associated pathways were top ranked KEGG pathways with high fold-change for low concentrations of 4-NQO and FA. The similar gene profiles perturbed by DCA with three test concentrations identified, the common GO BP terms associated with aromatic amino acid family biosynthetic process and ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway. DCA has no obvious genotoxicity as there was no enriched DNA damage and repair pathways and fold-change of replication and repair KEGG pathways were very low. Five genes (RAD18, RAD59, MUS81, MMS4, and BEM4) could serve as candidate genes for genotoxic chemicals. Overall, the yeast functional genomic profiling showed great performance for assessing the signatures and potential molecular mechanisms of genotoxic chemicals.

MiR-338-5p Levels and Cigarette Smoking are Associated With Neuropathic Pain Severity in Individuals With Spinal Cord Injury: Preliminary Findings From a Genome-Wide microRNA Expression Profiling Screen

ObjectiveTo identify microRNA biomarkers and clinical factors associated with neuropathic pain after spinal cord injury. DesignCross-sectional, secondary analysis of baseline data collected from ongoing clinical studies. Using a genome-wide microRNA screening approach, we studied differential microRNA expression in serum from 43 adults with spinal cord injury enrolled in ongoing clinical studies. Least squares regression was used to identify associations between microRNA expression, clinical factors, and neuropathic pain severity. SettingCommunity-dwelling individuals with spinal cord injury. ParticipantsParticipants (N=43) were at least 18 years old with spinal cord injury, with 28 reporting neuropathic pain and 15 reporting no neuropathic pain. InterventionsNot applicable. Main Outcome MeasuresPain presence, type, and intensity were assessed with the International Spinal Cord Injury Pain Basic Data Set. Serum microRNA normalized deep sequencing counts were quantified from blood samples. Participant demographic factors, injury characteristics, medication use, and health habits were collected via questionnaire. ResultsmiR-338-5p expression and history of cigarette smoking were associated with and explained 37% of the variance in neuropathic pain severity (R2=0.37, F2,18=5.31, P=.02) independent of other clinical factors. No association was identified between miR-338-5p levels and nociceptive pain severity. ConclusionsOur findings suggest that miR-338-5p and cigarette smoking may both play a role in the development or maintenance of neuropathic pain after spinal cord injury. While additional work is needed to confirm these findings, validated target analysis suggests a neuroprotective role of miR-338-5p in modulating neuroinflammation and neuronal apoptosis and that its downregulation may result in maladaptive neuroplastic mechanisms contributing to neuropathic pain after spinal cord injury.

Here and there: the double-side transgene localization.

Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identif ication of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact f iltration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s f lanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two f lanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the f ield of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to eff iciently localize genomic transposon integrations, which was conf irmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.

The effect of antioxidants in Ehrlich Ascites Cancer.

A fundamental goal in molecular oncology is to unravel the underlying mechanisms which cause the cell transformation. In line with this approach, genome-wide functional screening approaches have revealed exciting insights into heterogeneous nature of cancer. Rapidly expanding horizons of research have unraveled myriad of pathways which play instrumental role in carcinogenesis and metastasis. Oxidative stress has also been reported to be significantly involved in cancer onset and progression. In line with this approach, oxidative stress modulating chemicals have always been sharply divided into antioxidants and oxidative stress-inducing agents. Conceptual and experimental advancements have enabled us to critically analyze full potential of these two different groups of chemicals in cancer chemoprevention. Different antioxidants are currently being analyzed in different phases of clinical trials. Although it has been reported in the literature that antioxidant supplements reduce tumor cells in some tumors or cause volume reduction in solid tumor sizes, there is no definite consensus. Therefore, an antioxidant supplement guideline based on more detailed clinical research and as a result of these is needed to achieve the best care for cancer patients and to avoid risky treatments for cancer patients.

Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication.

ABSTRACTCellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1’s crucial role to the biogenesis of pan-flavivirus replication organelles.IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.

Hsa-MiR-19a-3p and hsa-MiR-19b-3p Are Associated with Spinal Cord Injury-Induced Neuropathic Pain: Findings from a Genome-Wide MicroRNA Expression Profiling Screen.

Neuropathic pain in spinal cord injury (SCI) is associated with inflammation in both the peripheral and central nervous system (CNS), which may contribute to the initiation and maintenance of persistent pain. An understanding of factors contributing to neuroinflammation may lead to new therapeutic targets for neuropathic pain. Moreover, novel circulating biomarkers of neuropathic pain may facilitate earlier and more effective treatment. MicroRNAs (miRNAs) are short, non-coding single-stranded RNA that have emerged as important biomarkers and molecular mediators in physiological and pathological conditions. Using a genome-wide miRNA screening approach, we studied differential miRNA expression in plasma from 68 healthy, community-dwelling adults with and without SCI enrolled in ongoing clinical studies. We detected 2367 distinct miRNAs. Of these, 383 miRNAs were differentially expressed in acute SCI or chronic SCI versus no SCI and 71 were differentially expressed in chronic neuropathic pain versus no neuropathic pain. We selected homo sapiens (hsa)-miR-19a-3p and hsa-miR-19b-3p for additional analysis based on p-value, fold change, and their known role as regulators of neuropathic pain and neuroinflammation. Both hsa-miR-19a-3p and hsa-miR-19b-3p levels were significantly higher in those with chronic SCI and severe neuropathic pain versus those with chronic SCI and no neuropathic pain. In confirmatory studies, both hsa-miR-19a-3p and hsa-miR-19b-3p have moderate to strong discriminative ability to distinguish between those with and without pain. After adjusting for opioid use, hsa-miR-19b-3p levels were positively associated with pain interference with mood. Because hsa-miR-19 levels have been shown to change in response to exercise, folic acid, and resveratrol, these studies suggest that miRNAs are potential targets of therapeutic interventions.

TRIMming viral infection: identification and characterization of the novel antiviral TRIM7

Abstract Noroviruses (NoVs) are the leading cause of acute viral gastroenteritis worldwide, causing up to 700 million infections and 200,000 deaths annually, and treatment is limited to supportive care as there are currently no targeted antivirals nor vaccines against NoVs. Therefore, it is critical to discover novel antivirals and develop targeted interventions in order to improve outcomes. Here we have used a novel genome-wide CRISPR activation screening approach to identify host genes that can inhibit murine norovirus (MNoV) infection or replication. A substantial number of high scoring gene candidates acted in interferon and immune regulation signaling networks, but several were not previously associated with antiviral activity or immunity. TRIM7, the strongest screen hit, was a little studied E3 ubiquitin ligase associated with metabolism and some cancers, yet its expression served as a powerful antiviral in an MNoV-infected cell. Since our initial screen, TRIM7 has begun to emerge as a strategic player in both positive and negative regulation of the innate immune response during infection. We have since characterized the antiviral mechanism of action of TRIM7 against MNoV in vitro, and have found TRIM7 to be acting early in the viral life cycle to limit viral protein production. Co-immunoprecipitation and in vitro viral evolution experiments have identified MNoV NS6 (viral protease) and NS7 (RNA-dependent RNA polymerase) as important targets of the antiviral activity of TRIM7, and also point to key residues of interest associated with MNoV resistance to TRIM7. Finally, we have investigated the relevance of TRIM7 during in vivo infection using knock-in mouse lines to determine tissue specificity and potency of the antiviral effect.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors.

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.