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

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

Can we forecast poor outcome in herpes simplex and varicella zoster encephalitis? A narrative review

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are among the most commonly diagnosed infectious causes of sporadic encephalitis worldwide. Despite treatment, mortality and morbidity rates remain high, especially for HSV encephalitis. This review is intended to provide an overview of the existing scientific literature on this topic from the perspective of a clinician who is confronted with serious decisions about continuation or withdrawal of therapeutic interventions. We performed a literature review searching two databases and included 55 studies in the review. These studies documented or investigated specifically outcome and predictive parameters of outcome of HSV and/or VZV encephalitis. Two reviewers independently screened and reviewed full-text articles meeting the inclusion criteria. Key data were extracted and presented as a narrative summary. Both, HSV and VZV encephalitis have mortality rates between 5 and 20% and complete recovery rates range from 14 to 43% for HSV and 33 to 49% for VZV encephalitis. Prognostic factors for both VZV and HSV encephalitis are older age and comorbidity, as well as severity of disease and extent of magnetic resonance imaging (MRI) lesions on admission, and delay in treatment initiation for HSV encephalitis. Although numerous studies are available, the main limiting factors are the inconsistent patient selection and case definitions as well as the non-standardised outcome measures, which hampers the comparability of the studies. Therefore, larger and standardised observational studies applying validated case definitions and outcome measures including quality of life assessment are required to provide solid evidence to answer the research question

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

SOX17 is a critical specifier of human primordial germ cell fate.

Specification of primordial germ cells (PGCs) marks the beginning of the totipotent state. However, without a tractable experimental model, the mechanism of human PGC (hPGC) specification remains unclear. Here, we demonstrate specification of hPGC-like cells (hPGCLCs) from germline competent pluripotent stem cells. The characteristics of hPGCLCs are consistent with the embryonic hPGCs and a germline seminoma that share a CD38 cell-surface marker, which collectively defines likely progression of the early human germline. Remarkably, SOX17 is the key regulator of hPGC-like fate, whereas BLIMP1 represses endodermal and other somatic genes during specification of hPGCLCs. Notable mechanistic differences between mouse and human PGC specification could be attributed to their divergent embryonic development and pluripotent states, which might affect other early cell-fate decisions. We have established a foundation for future studies on resetting of the epigenome in hPGCLCs and hPGCs for totipotency and the transmission of genetic and epigenetic information.We thank Rick Livesey and his lab for help with the culture of hESCs; Sohei Kitazawa and Janet Shipley for the TCam-2 cells; Nigel Miller and Andy Riddell for cell sorting, Roger Barker, Xiaoling He, and Pam Tyers for collection of human embryos; and Charles Bradshaw for help with bioinformatics. We thank members of the Surani and Hanna labs for important discussions and technical help. N.I. is supported by Grant-in-Aid for fellows of the JSPS and by BIRAX (the Britain Israel Research and Academic Exchange Partnership) initiative, who provided a project grant to J.H.H. and M.A.S. J.H.H. is supported by Ilana and Pascal Mantoux, the Kimmel Award, ERC (StG-2011-281906), Helmsley Charitable Trust, ISF (Bikura, Morasha, ICORE), ICRF, the Abisch Frenkel Foundation, the Fritz Thyssen Stiftung, Erica and Robert Drake, Benoziyo Endowment fund, and the Flight Attendant Medical Research Institute (FAMRI). J.H.H. is a New York Stem Cell Foundation Robertson Investigator. W.C.C.T. is supported by Croucher Foundation and Cambridge Trust; M.A.S. is supported by HFSP and a Wellcome Trust Investigator Award.This is the final version of the article, originally published in Cell, Volume 160, Issues 1-2, p253–268, 15 January 2015, doi: 10.1016/j.cell.2014.12.01

Epigenetic resetting of human pluripotency

Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, $XIST$-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.This research is funded by the Medical Research Council of the United Kingdom (MR/P00072X/1) and European Commission Framework 7 (HEALTH-F4-2013-602423, PluriMes), and in part by the UK Regenerative Medicine Platform (MR/L012537/1). WR is supported by the BBSRC (BB/K010867/1), Wellcome Trust (095645/Z/11/Z), EU BLUEPRINT, and EpiGeneSys. The Cambridge Stem Cell Institute receives core funding from the Wellcome Trust and the Medical Research Council. FvM was funded by a Postdoctoral Fellowship from the Swiss National Science Foundation (SNF)/Novartis. SM is funded by a Wellcome Trust PhD Studentship. AS is a Medical Research Council Professor

CCancer: a bird’s eye view on gene lists reported in cancer-related studies

CCancer is an automatically collected database of gene lists, which were reported mostly by experimental studies in various biological and clinical contexts. At the moment, the database covers 3369 gene lists extracted from 2644 papers published in ∼80 peer-reviewed journals. As input, CCancer accepts a gene list. An enrichment analyses is implemented to generate, as output, a highly informative survey over recently published studies that report gene lists, which significantly intersect with the query gene list. A report on gene pairs from the input list which were frequently reported together by other biological studies is also provided. CCancer is freely available at http://mips.helmholtz-muenchen.de/proj/ccancer

R spider: a network-based analysis of gene lists by combining signaling and metabolic pathways from Reactome and KEGG databases

R spider is a web-based tool for the analysis of a gene list using the systematic knowledge of core pathways and reactions in human biology accumulated in the Reactome and KEGG databases. R spider implements a network-based statistical framework, which provides a global understanding of gene relations in the supplied gene list, and fully exploits the Reactome and KEGG knowledge bases. R spider provides a user-friendly dialog-driven web interface for several model organisms and supports most available gene identifiers. R spider is freely available at http://mips.helmholtz-muenchen.de/proj/rspider

Murine malaria is associated with significant hearing impairment

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>malaria has been suspected to cause hearing loss. Developmental, cognitive and language disorders have been observed in children, surviving cerebral malaria. This prospective study aims to evaluate whether malaria influences hearing in mice.</p> <p>Methods</p> <p>Twenty mice were included in a standardized murine cerebral malaria model. Auditory evoked brainstem responses were assessed before infection and at the peak of the illness.</p> <p>Results</p> <p>A significant hearing impairment could be demonstrated in mice with malaria, especially the cerebral form. The control group did not show any alterations. No therapy was used.</p> <p>Conclusion</p> <p>This suggests that malaria itself leads to a hearing impairment in mice.</p

Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

Loss-of-function mutations in the cytosine-5 RNA methylase NSUN2 cause neurodevelopmental disorders in humans, yet the underlying cellular processes leading to the symptoms that include microcephaly remain unclear. Here, we show that NSUN2 is expressed in early neuroepithelial progenitors of the developing human brain, and its expression is gradually reduced during differentiation of human neuroepithelial stem (NES) cells in vitro. In the developing $\textit{Nsun2}$$^{-/-}$ mouse cerebral cortex, intermediate progenitors accumulate and upper-layer neurons decrease. Loss of NSUN2-mediated methylation of tRNA increases their endonucleolytic cleavage by angiogenin, and 5' tRNA fragments accumulate in $\textit{Nsun2}$$^{-/-}$ brains. Neural differentiation of NES cells is impaired by both $\textit{NSUN2}$ depletion and the presence of angiogenin. Since repression of NSUN2 also inhibited neural cell migration toward the chemoattractant fibroblast growth factor 2, we conclude that the impaired differentiation capacity in the absence of NSUN2 may be driven by the inability to efficiently respond to growth factors.This work was funded by Cancer Research UK (CR-UK C10701/A15181 ), Worldwide Cancer Research ( 15-0168 ), the Medical Research Council (MRC MR/M01939X/1 ), the European Research Council (ERC 310360 ), and EMBO. Research in M.F.'s laboratory was supported by a core support grant from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Cambridge Stem Cell Institute