Gregory Hannon

Provided is an improved design of shRNA based on struc tural mimics of miR-451 precursors. These miR-451 shRNA mimics are channeled through a novel small RNA biogen esis pathway, require AGO2 catalysis and are processed by Drosha but are independent of DICER processing. This miRNA pathway feeds active elements only into Ago2 because of its unique catalytic activity. These data demon strate that this newly identified small RNA biogenesis path way can be exploited in vivo to produce active molecules. Specification includes a Sequence Listing.

The emergence of CRISPR technologies has enabled rapid and precise gene editing opportunities. The Functional Genomics Centre (FGC) - a joint venture between Cancer Research UK’s (CRUK) drug discovery engine, Cancer Research Horizons, and AstraZeneca has been launched to better utilize and improve the CRISPR technology within cancer research. The FGC leverages the strengths of both CRUK’s and AstraZeneca’s world-class oncology research and drug discovery experience, with the potential to drive scientific innovation in how new drug targets are identified and validated, and uniquely democratize access to the latest cutting-edge research tools and expertise for many thousands of scientists and clinicians funded by CRUK. Here, we will showcase how the FGC is leading the development and refinement of functional genomics technologies. Through independent evaluation of publicly available …

Animal germ cells deploy a specialized small RNA-based silencing system, called the PIWI-interacting RNA (piRNA) pathway, to prevent unwanted expression of transposable elements and maintain genome integrity. In Drosophila germ cells, the majority of piRNA populations originate from dual-strand piRNA clusters, genomic regions highly enriched in transposon fragments, via an elaborate protein machinery centred on the heterochromatin protein 1 homolog, Rhino. Although Rhino binds to peptides carrying trimethylated H3K9 in vitro, it is not fully understood why in vivo only a fraction of H3K9me3-decorated heterochromatin is occupied by Rhino. Recent work uncovered that Rhino is recruited to a subset of piRNA clusters by the zinc finger protein Kipferl. Here we identify a Kipferl-independent mode of Rhino targeting that is dependent on the histone H3 lysine 27 methyltransferase Enhancer of Zeste and the presence of H3K9me3 and H3K27me3 marks. At Kipferl-independent sites, we find that Rhino, through its dimeric chromodomain, specifically binds to loci marked by both H3K9me3 and H3K27me3. These results expand our understanding of the characteristic binding profile of the heterochromatin protein Rhino. Our work reveals a role for dual histone modifications in defining the binding specificity of a chromatin protein.

Imaging mass cytometry (IMC) is a powerful technique capable of detecting over 30 markers on a single slide. It has been increasingly used for single-cell-based spatial phenotyping in a wide range of samples. However, it only acquires a rectangle field of view (FOV) with a relatively small size and low image resolution, which hinders downstream analysis. Here, we reported a highly practical dual-modality imaging method that combines high-resolution immunofluorescence (IF) and high-dimensional IMC on the same tissue slide. Our computational pipeline uses the whole-slide image (WSI) of IF as a spatial reference and integrates small-FOV IMC into a WSI of IMC. The high-resolution IF images enable accurate single-cell segmentation to extract robust high-dimensional IMC features for downstream analysis. We applied this method in esophageal adenocarcinoma of different stages, identified the single-cell …

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen of over 16,000 RNAi triggers against the SARS-CoV-2 genome, using a massively parallel assay to identify hyper-potent siRNAs. We selected Ten candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity (IC50 < 20 pM) and strong blockade of infectivity in live-virus experiments. We further enhanced this activity by combinatorial pairing of the siRNA candidates and identified cocktails that were active against multiple types of variants of concern (VOC). We then examined over 2,000 possible mutations in the siRNA target sites by using saturation mutagenesis and confirmed broad protection of the leading cocktail against future variants. Finally, we demonstrated that intranasal …

The present invention relates to a method of spatially barcoding a given location on a substrate, and further to spatially barcoding detection probes present in a sample such as a biological tissue specimen for the purposes of analysing molecular features present in the tissue. Such analysis may include: i) the spatial expression of one or more biological molecules, specifically; ii) the spatial analysis of the transcriptome and/or iii) the spatial analysis of the proteome, including post-translational protein modifications. The invention further relates to various component products for performing such methods that include reagents kits, instrumentation and software.

IntroductionVasculogenic mimicry (VM), the process of tumor cell trans-differentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including small cell lung cancer (SCLC). In genetically engineered mouse models (GEMMs) of SCLC, NOTCH and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs.MethodsWe analysed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX, GEMMs and patient biopsies. VM-proficient cell subpopulations in ex vivo cultures were molecularly …

We identify the Sodium Leak Channel Non-Selective Protein (NALCN) as a key regulator of cancer metastasis and non-malignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric (Prom1CreERT2/LacZ;KrasG12D;Trp53Flx/Flx; n=269), intestinal (Villin1-CreERT2;KrasG12D;Trp53Flx/Flx; n=141) or pancreatic adenocarcinomas (Pdx1-Cre;KrasG12D;Trp53Flx/+; n=55) in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice (Villin1-CreERT2;KrasG12D;Trp53Flx/Flx; n=28) with gadolinium–an imaging contrast agent and NALCN channel blocker–similarly increased CTCs and metastasis. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer(Prom1CreERT2/LacZ; n=174 …

Provided is an improved design of shRNA based on structural mimics of miR-451 precursors. These miR-451 shRNA mimics are channeled through a novel small RNA biogenesis pathway, require AGO2 catalysis and are processed by Drosha but are independent of DICER processing. This miRNA pathway feeds active elements only into Ago2 because of its unique catalytic activity. These data demonstrate that this newly identified small RNA biogenesis pathway can be exploited in vivo to produce active molecules.

Pseudo-vascular network formation capacity in vitro is considered a key characteristic of vasculogenic mimicry. While many cancer cell lines are known to form pseudo-vascular networks, little is known about the spatiotemporal dynamics of these formations. Here, we present a framework for monitoring and characterising the dynamic formation and dissolution of pseudo-vascular networks in vitro. The framework combines time-resolved optical microscopy with open-source image analysis for network feature extraction and statistical modelling. The framework is demonstrated by comparing diverse cancer cell lines associated with vasculogenic mimicry, then in detecting response to drug compounds proposed to affect formation of vasculogenic mimics. Dynamic datasets collected were analysed morphometrically and a descriptive statistical analysis model was developed in order to measure stability and dissimilarity characteristics of the pseudo-vascular networks formed. Melanoma cells formed the most stable pseudo-vascular networks and were selected to evaluate the response of their pseudo-vascular networks to treatment with axitinib, brucine and tivantinib. Our framework is shown to enable quantitative analysis of both the capacity for network formation, linked vasculogenic mimicry, as well as dynamic responses to treatment.

Vasculogenic mimicry (VM) describes the formation of pseudo blood vessels constructed of tumor cells that have acquired endothelial-like properties. VM channels endow the tumor with a tumor-derived vascular system that directly connects to host blood vessels, and their presence is generally associated with poor patient prognosis. Here we show that the transcription factor, Foxc2, promotes VM in diverse solid tumor types by driving ectopic expression of endothelial genes in tumor cells, a process that is stimulated by hypoxia. VM-proficient tumors are resistant to anti-angiogenic therapy, and suppression of Foxc2 augments response. This work establishes co-option of an embryonic endothelial transcription factor by tumor cells as a key mechanism driving VM proclivity and motivates the search for VM-inhibitory agents that could form the basis of combination therapies with anti-angiogenics.

Spatial omics has been widely heralded as the new frontier in life sciences. This term encompasses a wide range of techniques that promise to transform many areas of biology and eventually revolutionize pathology by measuring physical tissue structure and molecular characteristics at the same time. Although the field came of age in the past 5 years, it still suffers from some growing pains: barriers to entry, robustness, unclear best practices for experimental design and analysis, and lack of standardization. In this Review, we present a systematic catalog of the different families of spatial omics technologies; highlight their principles, power, and limitations; and give some perspective and suggestions on the biggest challenges that lay ahead in this incredibly powerful—but still hard to navigate—landscape.

The PIWI-interacting RNA (piRNA) pathway prevents endogenous genomic parasites, i.e. transposable elements, from damaging the genetic material of animal gonadal cells. Specific regions in the genome, called piRNA clusters, are thought to define each species’ piRNA repertoire and therefore its capacity to recognize and silence specific transposon families. The unistrand cluster flamenco (flam) is essential in the somatic compartment of the Drosophila ovary to restrict Gypsy-family transposons from infecting the neighbouring germ cells. Disruption of flam results in transposon de-repression and sterility, yet it remains unknown whether this silencing mechanism is present more widely. Here, we systematically characterise 119 Drosophila species and identify five additional flam-like clusters separated by up to 45 million years of evolution. Small RNA-sequencing validated these as bona-fide unistrand piRNA …

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen with over 16,000 RNAi triggers against the SARS-CoV-2 genome using a massively parallel assay to identify hyper-potent siRNAs. We selected 10 candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity with IC50< 20pM and strong neutralisation in live virus experiments. We further enhanced the activity by combinatorial pairing of the siRNA candidates to develop siRNA cocktails and found that these cocktails are active against multiple types of variants of concern (VOC). We examined over 2,000 possible mutations to the siRNA target sites using saturation mutagenesis and identified broad protection against future variants. Finally, we demonstrated that intranasal administration of the …

PIWI-interacting RNAs (piRNAs) are small RNAs bound by PIWI-clade Argonaute proteins that function to silence transposable elements (TEs). Following mouse primordial germ cell (mPGC) specification around E6.25, fetal piRNAs emerge in male gonocytes from E13.5 onward. The in vitro differentiation of mPGC-like cells (mPGCLCs) has raised the possibility of studying the fetal piRNA pathway in greater depth. However, using single-cell RNA-seq and RT-qPCR along mPGCLC differentiation, we find that piRNA pathway factors are not fully expressed in Day 6 mPGCLCs. Moreover, we do not detect piRNAs across a panel of Day 6 mPGCLC lines using small RNA-seq. Our combined efforts highlight that in vitro differentiated Day 6 mPGCLCs do not yet resemble E13.5 or later mouse gonocytes where the piRNA pathway is active. This Matters Arising paper is in response to von Meyenn et al. (2016), published in …

Ductal carcinoma in situ (DCIS) is considered a non-invasive precursor to breast cancer, and although associated with an increased risk of developing invasive disease, many women with DCIS will never progress beyond their in situ diagnosis. The path from normal duct to invasive disease is not well understood, and efforts to do so are hampered by the substantial heterogeneity that exists between patients and even within patients. Using gene expression analysis, we have generated a ‘Timeline’ of disease progression, utilising the variability within patients and combining >2,000 individually micro-dissected ductal lesions from 145 patients into one continuous trajectory. Using this Timeline we show there is a progressive loss in basal layer integrity, coupled with two epithelial to mesenchymal transitions (EMT), one early in the timeline and a second just prior to cells leaving the duct. We identify early processes and potential biomarkers, including CAMK2N1, MNX1, ADCY5, HOXC11 and ANKRD22, whose reduced expression is associated with the progression of DCIS to invasive breast cancer.

We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium—a NALCN channel blocker—similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates …

PHGDH heterogeneity potentiates cancer cell dissemination and metastasis (vol 605. pg 747, 2022) logo search Research Study About KU Leuven Download PDF Nature Publication date: 2022-09-15 Volume: 609 Pages: E8 - E8 DOI: 10.1038/s41586-022-05226-7 Publisher: Nature Research PHGDH heterogeneity potentiates cancer cell dissemination and metastasis (vol 605. pg 747, 2022) Author: Rossi, Matteo Altea-Manzano, Patricia ; Demicco, Margherita ; Doglioni, Ginevra ; Bornes, Laura ; Fukano, Marina ; Vandekeere, Anke ; Cuadros, Alejandro M ; Fernandez-Garcia, Juan ; Riera-Domingo, Carla ; Jauset, Cristina ; Planque, Melanie ; Alkan, H Furkan ; Nittner, David ; Zuo, Dongmei ; Broadfield, Lindsay A ; Parik, Sweta ; Pane, Antonino Alejandro ; Rizzollo, Francesca ; Rinaldi, Gianmarco ; Zhang, Tao ; Teoh, Shao Thing ; Aurora, Arin B ; Karras, Panagiotis ; Vermeire, Ines ; Broekaert, Dorien ; Van Elsen, …

Ductal carcinoma in situ (DCIS) is considered a non-invasive precursor to breast cancer, and although associated with an increased risk of developing invasive disease, many women with DCIS will never progress beyond their in situ diagnosis. The path from normal duct to invasive ductal carcinoma (IDC) is not well understood, and efforts to do so are hampered by the substantial heterogeneity that exists between patients, and even within patients. Here we show gene expression analysis from > 2,000 individually micro-dissected ductal lesions representing 145 patients. Combining all samples into one continuous trajectory we show there is a progressive loss in basal layer integrity heading towards IDC, coupled with two epithelial to mesenchymal transitions, one early and a second coinciding with the convergence of DCIS and IDC expression profiles. We identify early processes and potential biomarkers …

PIWI-interacting RNAs (piRNAs) are small RNAs required to recognize and silence transposable elements. The 5’ ends of mature piRNAs are defined through cleavage of long precursor transcripts, primarily by Zucchini (Zuc). Zuc-dependent cleavage typically occurs immediately upstream of a uridine. However, Zuc lacks sequence preference in vitro, pointing towards additional unknown specificity factors. Here, we examine murine piRNAs and reveal a strong and specific enrichment of three sequences (UAA, UAG, UGA)—corresponding to stop codons—at piRNA 5’ ends. Stop codon sequences are also enriched immediately after piRNA processing intermediates, reflecting their Zuc-dependent tail-to-head arrangement. Further analyses reveal that a Zuc in vivo cleavage preference at four sequences (UAA, UAG, UGA, UAC) promotes 5’ end stop codons. This observation is conserved across mammals and …

Tumour heterogeneity is thought to be a major barrier to successful cancer treatment due to the presence of drug resistant clonal lineages. However, identifying the characteristics of such lineages that underpin resistance to therapy has remained challenging. Here, we utilise clonal transcriptomics with WILD-seq; Wholistic Interrogation of Lineage Dynamics by sequencing, in mouse models of triple-negative breast cancer (TNBC) to understand response and resistance to therapy, including BET bromodomain inhibition and taxane-based chemotherapy. These analyses revealed oxidative stress protection by NRF2 as a major mechanism of taxane resistance and led to the discovery that our tumour models are collaterally sensitive to asparagine deprivation therapy using the clinical stage drug L-asparaginase after frontline treatment with docetaxel. In summary, clonal transcriptomics with WILD-seq identifies mechanisms of resistance to chemotherapy that are also operative in patients and pin points asparagine bioavailability as a druggable vulnerability of taxane-resistant lineages.

A set of increasingly powerful approaches are enabling spatially resolved measurements of growing numbers of molecular features in biological samples. While important insights can be derived from the two-dimensional data that many of these technologies generate, it is clear that extending these approaches into the third and fourth dimensions will magnify their impact. Realizing biological insights from datasets where thousands to millions of cells are annotated with tens to hundreds of parameters in space will require the development of new computational and visualization strategies. Here, we describe Theia, a virtual reality-based platform, which enables exploration and analysis of either volumetric or segmented, molecularly-annotated, three-dimensional datasets, with the option to extend the analysis to time-series data. We also describe our pipeline for generating annotated 3D models of breast cancer and supply several datasets to enable users to explore the utility of Theia for understanding cancer biology in three dimensions.

Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs. Genetic, transcriptional and translational intra-tumor heterogeneity contributes to this dynamic process. Beyond this, metabolic intra-tumor heterogeneity has also been observed, yet its role for cancer progression remains largely elusive. Here, we discovered that intra-tumor heterogeneity in phosphoglycerate dehydrogenase (PHGDH) protein expression drives breast cancer cell dissemination and metastasis formation. Specifically, we observed intra-tumor heterogeneous PHGDH expression in primary breast tumors, with low PHGDH expression being indicative of metastasis in patients. In mice, Phgdh protein, but not mRNA, expression is low in circulating tumor cells and early metastatic lesions, leading to increased dissemination and metastasis formation. Mechanistically, low PHGDH protein expression induces an imbalance in glycolysis that can activate sialic acid synthesis. Consequently, cancer cells undergo a partial EMT and show increased p38 as well as SRC phosphorylation, which activate cellular programs of dissemination. In turn, inhibition of sialic acid synthesis through knock-out of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) counteracts the increased cancer cell dissemination and metastasis induced by low PHGDH expression. In conclusion, we find that heterogeneity in PHGDH protein expression promotes cancer cell dissemination and metastasis formation.

With the aim of producing a 3D representation of tumours, IMAXT uses a large variety of modalities in order to acquire tumour samples and produce a map of every cell in the tumour and its host environment. With the large volume and variety of data produced in the project we develop automatic data workflows and analysis pipelines and introduce a research methodology where scientists connect to a cloud environment to perform analysis close to where data are located instead of bringing data to their local computers. Here we present the data and analysis infrastructure, discuss the unique computational challenges and describe the analysis chains developed and deployed to generate molecularly annotated tumour models. Registration is achieved by use of a novel technique involving spherical fiducial marks that are visible in all imaging modalities used within IMAXT.

This invention relates to a novel linker compound of formula (I) disclosed herein. The invention also relates to antibody conjugates. The invention relates to methods which utilise the compounds and conjugates disclosed herein.

Despite current advancements in research and therapeutics, lung cancer remains the leading cause of cancer-related mortality worldwide. This is mainly due to the resistance that patients develop against chemotherapeutic agents over the course of treatment. In the context of non-small cell lung cancers (NSCLC) harboring EGFR-oncogenic mutations, augmented levels of AXL and GAS6 have been found to drive resistance to EGFR tyrosine kinase inhibitors such as Erlotinib and Osimertinib in certain tumors with mesenchymal-like features. By studying the ontogeny of AXL-positive cells, we have identified a novel non-genetic mechanism of drug resistance based on cell-state transition. We demonstrate that AXL-positive cells are already present as a subpopulation of cancer cells in Erlotinib-naïve tumors and tumor-derived cell lines and that the expression of AXL is regulated through a stochastic mechanism centered on the epigenetic regulation of miR-335. The existence of a cell-intrinsic program through which AXL-positive/Erlotinib-resistant cells emerge infers the need of treating tumors harboring EGFR-oncogenic mutations upfront with combinatorial treatments targeting both AXL-negative and AXL-positive cancer cells.

The nuclear pore complex (NPC) is the principal gateway between nucleus and cytoplasm that enables exchange of macromolecular cargo. Composed of multiple copies of ~30 different nucleoporins (Nups), the NPC acts as a selective portal, interacting with factors which individually license passage of specific cargo classes. Here we show that two Nups of the inner channel, Nup54 and Nup58, are essential for transposon silencing via the PIWI-interacting RNA (piRNA) pathway in the Drosophila ovary. In ovarian follicle cells, loss of Nup54 and Nup58 results in compromised piRNA biogenesis exclusively from the flamenco locus, whereas knockdowns of other NPC subunits have widespread consequences. This provides evidence that some Nups can acquire specialised roles in tissue-specific contexts. Our findings consolidate the idea that the NPC has functions beyond simply constituting a barrier to nuclear/cytoplasmic exchange as genomic loci subjected to strong selective pressure can exploit NPC subunits to facilitate their expression.

Abstract The PIWI-interacting RNA (piRNA) pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. In Drosophila, piRNAs are intergenerationally inherited through the maternal lineage, and this has demonstrated importance in the specification of piRNA source loci and in silencing of I-and P-elements in the germ cells of daughters. Maternally inherited Piwi protein enters somatic nuclei in early embryos prior to zygotic genome activation and persists therein for roughly half of the time required to complete embryonic development. To investigate the role of the piRNA pathway in the embryonic soma, we created a conditionally unstable Piwi protein. This enabled maternally deposited Piwi to be cleared from newly laid embryos within 30 min and well ahead of the activation of zygotic transcription. Examination of RNA and protein profiles over time, and correlation with patterns of H3K9me3 deposition, suggests a role for maternally deposited Piwi in attenuating zygotic transposon expression in somatic cells of the developing embryo. In particular, robust deposition of piRNAs targeting roo, an element whose expression is mainly restricted to embryonic development, results in the deposition of transient heterochromatic marks at active roo insertions. We hypothesize that roo, an extremely successful mobile element, may have adopted a lifestyle of expression in the embryonic soma to evade silencing in germ cells.

The heterogeneity of breast cancer plays a major role in drug response and resistance and has been extensively characterized at the genomic level. Here, a single-cell breast cancer mass cytometry (BCMC) panel is optimized to identify cell phenotypes and their oncogenic signalling states in a biobank of patient-derived tumour xenograft (PDTX) models representing the diversity of human breast cancer. The BCMC panel identifies 13 cellular phenotypes (11 human and 2 murine), associated with both breast cancer subtypes and specific genomic features. Pre-treatment cellular phenotypic composition is a determinant of response to anticancer therapies. Single-cell profiling also reveals drug-induced cellular phenotypic dynamics, unravelling previously unnoticed intra-tumour response diversity. The comprehensive view of the landscapes of cellular phenotypic heterogeneity in PDTXs uncovered by the BCMC panel …

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine (NE) cancer with rapid and widespread metastasis and a dismal prognosis measurable only in months. The inherent lack of tumor biopsies has limited our understanding of SCLC phenotypic heterogeneity, and except for the addition of Immune Checkpoint Inhibitors the therapeutic strategy has remained largely unchanged for decades. We are investigating Vascular Mimicry (VM) in SCLC, which is linked to poor prognosis and may provide a novel therapeutic opportunity1. VM describes the ability of aggressive tumor cells to undergo trans-endothelial differentiation, enabling angiogenesis-independent tumor vascularization. We hypothesize that, via extracellular matrix (ECM) remodeling, VM provides a favorable tumor microenvironment (TME) to support tumor progression and metastasis.We explored SCLC VM competency and molecular …

In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex.

Background Partially methylated domains (PMDs) are a hallmark of epigenomes in reproducible and specific biological contexts, including cancer cells, the placenta, and cultured cell lines. Existing methods for deciding whether PMDs exist in a sample, as well as their identification, are few, often tailored to specific biological questions, and require high coverage samples for accurate identification. Results In this study, we outline a set of axioms that take a step towards a functional definition for PMDs, describe an improved method for comparable PMD detection across samples with substantially differing sequencing depths, and refine the decision criteria for whether a sample contains PMDs using a data-driven approach. Applying our method to 267 methylomes from 7 species, we corroborated recent results regarding the general association between replication timing and PMD state, and report identification of …

The present invention provides a method for targeted cell retrieval, comprising: providing a population of barcoded cells, said population comprising a plurality of different barcodes, each of the plurality of different barcodes being uniquely targetable with a target-specific CRISPR RNA; introducing a CRISPR-Cas system, or one or more vectors encoding the components of the CRISPR-Cas system, into the population of barcoded cells said CRISPR-Cas system having a target-specific CRISPR RNA that targets a first barcode of said plurality of different barcodes, thereby causing a CRISPR-Cas system-mediated change at a target site leading to a change in one or more detectable properties of at least one cell carrying said first barcode; and retrieving said at least one cell carrying said first barcode based on the change in said one or more detectable properties. Also provided are products and kits for use in the method …

Previously we showed that 31/1,032 (3%) asymptomatic healthcare workers (HCW) from a large teaching hospital in Cambridge UK tested positive for SARS-CoV-2 in April 2020. 26/169 (15%) HCWs with symptoms of coronavirus disease 2019 (COVID-19) also tested positive (Rivett et al., 2020). Here we show that the proportion of both asymptomatic and symptomatic HCWs testing positive rapidly declined to near-zero between 25th April and 24th May 2020, corresponding with a decline in patient admissions with COVID-19 during the ongoing UK 'lockdown'. These data demonstrate how infection prevention and control measures including staff testing may help prevent hospitals from becoming independent 'hubs' of SARS-CoV-2 transmission, and illustrate how, with appropriate precautions, organisations in other sectors may be able to resume on-site work safely.

GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by …