Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids

Approaches based on genetic modification have been invaluable for investigating a wide array of biological processes, with gain- and loss-of-function approaches frequently used to investigate gene function. However, the presence of paralogues, and hence possible genetic compensation, for many genes necessitates the knockout (KO) of all paralogous genes in order to observe clear phenotypic change. CRISPR technology, the most recently described tool for gene editing, can generate KOs with unprecedented ease and speed and has been used in adult stem cell-derived organoids for single gene knockout, gene knock-in and gene correction. However, the simultaneous targeting of multiple genes in organoids by CRISPR technology has not previously been described. Here we describe a rapid, scalable and cost effective method for generating double knockouts in organoids. By concatemerizing multiple gRNA expression cassettes, we generated a ‘gRNA concatemer vector’. Our method allows the rapid assembly of annealed synthetic DNA oligos into the final vector in a single step. This approach facilitates simultaneous delivery of multiple gRNAs to allow up to 4 gene KO in one step, or potentially to increase the efficiency of gene knockout by providing multiple gRNAs targeting one gene. As a proof of concept, we knocked out negative regulators of the Wnt pathway in small intestinal organoids, thereby removing their growth dependence on the exogenous Wnt enhancer, R-spondin1.A.A-R. is supported by the Medical Research Council (MRC), A.M.is supported by Wntsapp (Marie Curie ITN) and B-K.K. and R.M. are supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [101241/Z/13/Z] and receive support through a core grant from the Wellcome Trust and MRC to the WT-MRC Cambridge Stem Cell Institute

Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids

Human embryonic stem cells cultured in 2D micropatterns with BMP4 differentiate into a radial arrangement of germ layers and extraembryonic cells. Single-cell transcriptomes demonstrate generation of cell types transcriptionally similar to their in vivo counterparts in Carnegie stage 7 human gastrula. Time-course analyses indicate sequential differentiation, where the epiblast arises by 12 h between the prospective ectoderm in the center and the cells initiating differentiation toward extraembryonic fates at the edge. Extraembryonic and mesendoderm precursors arise from the epiblast by 24 h, while nascent mesoderm, endoderm, and primordial germ cell-like cells form by 44 h. Dynamic changes in transcripts encoding signaling components support a BMP, WNT, and Nodal hierarchy underlying germ-layer specification conserved across mammals, and FGF and HIPPO pathways being active throughout differentiation. This work also provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation

Cryptococcal transmigration across a model brain blood-barrier: Evidence of the Trojan horse mechanism and differences between Cryptococcus neoformans var. grubii strain H99 and Cryptococcus gattii strain R265

© 2015 Institut Pasteur. Cryptococcus neoformans (. Cn) and Cryptococcus gattii (Cg) cause neurological disease and cross the BBB as free cells or in mononuclear phagocytes via the Trojan horse mechanism, although evidence for the latter is indirect. There is emerging evidence that Cn and the North American outbreak Cg strain (R265) more commonly cause neurological and lung disease, respectively. We have employed a widely validated in vitro model of the BBB, which utilizes the hCMEC/D3 cell line derived from human brain endothelial cells (HBEC) and the human macrophage-like cell line, THP-1, to investigate whether transport of dual fluorescence-labelled Cn and Cg across the BBB occurs within macrophages. We showed that phagocytosis of Cn by non-interferon (IFN)-γ stimulated THP-1 cells was higher than that of Cg. Although Cn and Cg-loaded THP-1 bound similarly to TNF-activated HBECs under shear stress, more Cn-loaded macrophages were transported across an intact HBEC monolayer, consistent with the predilection of Cn for CNS infection. Furthermore, Cn exhibited a higher rate of expulsion from transmigrated THP-1 compared with Cg. Our results therefore provide further evidence for transmigration of both Cn and Cg via the Trojan horse mechanism and a potential explanation for the predilection of Cn to cause CNS infection

Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development.

Early mouse development is accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2), which is essential for embryonic development. Here we show that genome-wide accumulation of H3K9me2 is crucial for postimplantation development, and coincides with redistribution of enhancer of zeste homolog 2 (EZH2)-dependent histone H3 lysine 27 trimethylation (H3K27me3). Loss of G9a or EZH2 results in upregulation of distinct gene sets involved in cell cycle regulation, germline development and embryogenesis. Notably, the H3K9me2 modification extends to active enhancer elements where it promotes developmentally-linked gene silencing and directly marks promoters and gene bodies. This epigenetic mechanism is important for priming gene regulatory networks for critical cell fate decisions in rapidly proliferating postimplantation epiblast cells.Wellcome Trust: Jan J Zylicz, Ufuk Günesdogan, Jamie A Hackett, Delphine Cougot, Caroline Lee, MA Surani, WT096738; European Commission (EC): Ufuk Günesdogan; Wellcome Trust: Jan J Zylicz, RG44593This is the final version of the article. It was first available from eLife via http://dx.doi.org/10.7554/eLife.0957

High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human ESC gastruloid cultures

During mammalian gastrulation, germ layers arise and are shaped into the body plan while extraembryonic layers sustain the embryo. Human embryonic stem cells, cultured with BMP4 on extracellular matrix micro-discs, reproducibly differentiate into gastruloids, expressing markers of germ layers and extraembryonic cells in radial arrangement. Using single-cell RNA sequencing and cross-species comparisons with mouse, cynomolgus monkey gastrulae, and post-implantation human embryos, we reveal that gastruloids contain cells transcriptionally similar to epiblast, ectoderm, mesoderm, endoderm, primordial germ cells, trophectoderm, and amnion. Upon gastruloid dissociation, single cells reseeded onto micro-discs were motile and aggregated with the same but segregated from distinct cell types. Ectodermal cells segregated from endodermal and extraembryonic but mixed with mesodermal cells. Our work demonstrates that the gastruloid system models primate-specific features of embryogenesis, and that gastruloid cells exhibit evolutionarily conserved sorting behaviors. This work generates a resource for transcriptomes of human extraembryonic and embryonic germ layers differentiated in a stereotyped arrangement

A conceptual and computational framework for modelling and understanding the non-equilibrium gene regulatory networks of mouse embryonic stem cells

The capacity of pluripotent embryonic stem cells to differentiate into any cell type in the body makes them invaluable in the field of regenerative medicine. However, because of the complexity of both the core pluripotency network and the process of cell fate computation it is not yet possible to control the fate of stem cells. We present a theoretical model of stem cell fate computation that is based on Halley and Winkler's Branching Process Theory (BPT) and on Greaves et al.'s agent-based computer simulation derived from that theoretical model. BPT abstracts the complex production and action of a Transcription Factor (TF) into a single critical branching process that may dissipate, maintain, or become supercritical. Here we take the single TF model and extend it to multiple interacting TFs, and build an agent-based simulation of multiple TFs to investigate the dynamics of such coupled systems. We have developed the simulation and the theoretical model together, in an iterative manner, with the aim of obtaining a deeper understanding of stem cell fate computation, in order to influence experimental efforts, which may in turn influence the outcome of cellular differentiation. The model used is an example of self-organization and could be more widely applicable to the modelling of other complex systems. The simulation based on this model, though currently limited in scope in terms of the biology it represents, supports the utility of the Halley and Winkler branching process model in describing the behaviour of stem cell gene regulatory networks. Our simulation demonstrates three key features: (i) the existence of a critical value of the branching process parameter, dependent on the details of the cistrome in question; (ii) the ability of an active cistrome to "ignite" an otherwise fully dissipated cistrome, and drive it to criticality; (iii) how coupling cistromes together can reduce their critical branching parameter values needed to drive them to criticality.This work was performed as part of the CellBranch project, funded by the UK’s Biotechnology and Biological Sciences Research Council (BBSRC, http://www.bbsrc.ac.uk/), project reference BB/L018705/1. Grant holder: SS

Activation of lineage-regulators and transposable elements across a pluripotent spectrum

Embryonic stem cells (ESC) are characterised by the pluripotent capacity to generate all embryonic lineages. Here we show ESC can occupy a spectrum of distinct transcriptional and epigenetic states in response to varied extrinsic conditions. This spectrum broadly corresponds to a developmental continuum of pluripotency and is coupled with a gradient of increasing global DNA methylation. Each pluripotent state is linked with activation of distinct classes of transposable elements (TE), which in turn influence ESC through generating chimeric transcripts. Moreover, varied ESC culture parameters differentially license heterogeneous activation of master lineageregulators, including Sox1, Gata4 or Blimp1, and influence differentiation. Activation of Blimp1 is prevalent in 2i (without-LIF) conditions, and marks a dynamic primordial germ cell (PGC)-like sub-state, that is directly repressed by Klf4 downstream of LIF/STAT3 signalling. Thus, extrinsic cues establish a spectrum of pluripotent states, in part by modulating sub-populations, as well as directing the transcriptome, epigenome, and TE.Funding for this study came from a Wellcome Trust program grant (to M.A.S.), and Cancer Research UK (C6946/A14492)/Wellcome Trust (092096) core grants

Nodding syndrome in Tanzania may not be associated with circulating anti-NMDA- and anti-VGKC receptor antibodies or decreased pyridoxal phosphate serum levels-a pilot study

Background: Nodding syndrome (NS) is a seemingly progressive epilepsy disorder of unknown underlying cause. We investigated association of pyridoxal-phosphate serum levels and occurrence of anti-neuronal antibodies against N-methyl-D-aspartate (NMDA) receptor and voltage gated potassium channel (VGKC) complex in NS patients.Methods: Sera of a Tanzanian cohort of epilepsy and NS patients and community controls were tested for the presence of anti-NMDA-receptor and anti-VGKC complex antibodies by indirect immunofluorescence assay. Furthermore pyridoxal-phosphate levels were measured.Results: Auto-antibodies against NMDA receptor or VGKC (LG1 or Caspr2) complex were not detected in sera of patients suffering from NS (n=6), NS plus other seizure types (n=16), primary generalized epilepsy (n=1) and community controls without epilepsy (n=7). Median Pyridoxal-phosphate levels in patients with NS compared to patients with primary generalized seizures and community controls were not significantly different. However, these median pyridoxal-phosphate levels are significantly lower compared to the range considered normal in Europeans.Conclusions: In this pilot study NS was not associated with serum anti-NMDA receptor or anti-VGKC complex antibodies and no association to pyridoxal-phosphate serum levels was found.Key words: nodding syndrome, epilepsy, anti-neuronal antibodies, pyridoxal-phosphat

Loss of 5-methylcytosine alters the biogenesis of vault-derived small RNAs to coordinate epidermal differentiation.

The presence and absence of RNA modifications regulates RNA metabolism by modulating the binding of writer, reader, and eraser proteins. For 5-methylcytosine (m5C) however, it is largely unknown how it recruits or repels RNA-binding proteins. Here, we decipher the consequences of m5C deposition into the abundant non-coding vault RNA VTRNA1.1. Methylation of cytosine 69 in VTRNA1.1 occurs frequently in human cells, is exclusively mediated by NSUN2, and determines the processing of VTRNA1.1 into small-vault RNAs (svRNAs). We identify the serine/arginine rich splicing factor 2 (SRSF2) as a novel VTRNA1.1-binding protein that counteracts VTRNA1.1 processing by binding the non-methylated form with higher affinity. Both NSUN2 and SRSF2 orchestrate the production of distinct svRNAs. Finally, we discover a functional role of svRNAs in regulating the epidermal differentiation programme. Thus, our data reveal a direct role for m5C in the processing of VTRNA1.1 that involves SRSF2 and is crucial for efficient cellular differentiation.We thank everybody who provided us with reagents, in particular we thank James Stevenin for sending us recombinant SRSF2. We gratefully acknowledge the support of all the WT-MRC Stem Cell Institute core facility managers. This work was funded by Cancer Research UK (CR-630 UK) and the European Research Council (ERC). Parts of this research in Michaela Frye's laboratory was supported by core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute. Juri Rappsilber’s laboratory was supported by Wellcome Trust Senior Research Fellowship (084229). Gracjan Michlewski’s laboratory was supported by the MRC Career Development Award (G10000564), Wellcome Trust Seed Award (210144/Z/18/Z) and Wellcome Trust Centre for Cell Biology Core Grants (077707 and 092076). Abdulrahim Sajini was supported by a scholarship from the University of Tabuk and Khalifa University of Science and Technology Faculty start-up award number FSU-2018-01. Rebecca Wagner was supported by the Wellcome Trust PhD Programme in Stem Cell Biology & Medicine