Publications by authors named "William C Skarnes"

62 Publications

Generation and basic characterization of a gene-trap knockout mouse model of Scn2a with a substantial reduction of voltage-gated sodium channel Na 1.2 expression.

Genes Brain Behav 2020 Dec 25:e12725. Epub 2020 Dec 25.

Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, Indiana, USA.

Large-scale genetic studies revealed SCN2A as one of the most frequently mutated genes in patients with neurodevelopmental disorders. SCN2A encodes for the voltage-gated sodium channel isoform 1.2 (Na 1.2) expressed in the neurons of the central nervous system. Homozygous knockout (null) of Scn2a in mice is perinatal lethal, whereas heterozygous knockout of Scn2a (Scn2a ) results in mild behavior abnormalities. The Na 1.2 expression level in Scn2a mice is reported to be around 50-60% of the wild-type (WT) level, which indicates that a close to 50% reduction of Na 1.2 expression may not be sufficient to lead to major behavioral phenotypes in mice. To overcome this barrier, we characterized a novel mouse model of severe Scn2a deficiency using a targeted gene-trap knockout (gtKO) strategy. This approach produces viable homozygous mice (Scn2a ) that can survive to adulthood, with about a quarter of Na 1.2 expression compared to WT mice. Innate behaviors like nesting and mating were profoundly disrupted in Scn2a mice. Notably, Scn2a mice have a significantly decreased center duration compared to WT in the open field test, suggesting anxiety-like behaviors in a novel, open space. These mice also have decreased thermal and cold tolerance. Additionally, Scn2a mice have increased fix-pattern exploration in the novel object exploration test and a slight increase in grooming, indicating a detectable level of repetitive behaviors. They bury little to no marbles and have decreased interaction with novel objects. These Scn2a gene-trap knockout mice thus provide a unique model to study pathophysiology associated with severe Scn2a deficiency.
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http://dx.doi.org/10.1111/gbb.12725DOI Listing
December 2020

A Novel Chemically Differentiated Mouse Embryonic Stem Cell-Based Model to Study Liver Stages of Plasmodium berghei.

Stem Cell Reports 2020 06 21;14(6):1123-1134. Epub 2020 May 21.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK; Molecular Infection Medicine Sweden and Molecular Biology Department, Umeå University, 90187 Umeå, Sweden. Electronic address:

Asymptomatic and obligatory liver stage (LS) infection of Plasmodium parasites presents an attractive target for antimalarial vaccine and drug development. Lack of robust cellular models to study LS infection has hindered the discovery and validation of host genes essential for intrahepatic parasite development. Here, we present a chemically differentiated mouse embryonic stem cell (ESC)-based LS model, which supports complete development of Plasmodium berghei exoerythrocytic forms (EEFs) and can be used to define new host-parasite interactions. Using our model, we established that host Pnpla2, coding for adipose triglyceride lipase, is dispensable for P. berghei EEF development. In addition, we also evaluated in-vitro-differentiated human hepatocyte-like cells (iHLCs) to study LS of P. berghei and found it to be a sub-optimal infection model. Overall, our results present a new mouse ESC-based P. berghei LS infection model that can be utilized to study the impact of host genetic variation on parasite development.
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http://dx.doi.org/10.1016/j.stemcr.2020.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355138PMC
June 2020

Loss of IL-10 signaling in macrophages limits bacterial killing driven by prostaglandin E2.

J Exp Med 2020 02;217(2)

Translational Gastroenterology Unit, Experimental Medicine Division, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB-/- iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB-/- Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB-/- Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.
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http://dx.doi.org/10.1084/jem.20180649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041704PMC
February 2020

Improving homology-directed repair efficiency in human stem cells.

Methods 2019 07 16;164-165:18-28. Epub 2019 Jun 16.

The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA.

The generation of induced pluripotent stem cell models of human disease requires efficient modification of one or both alleles depending on dominant or recessive inheritance of the disease. To faithfully recapitulate many disease variants, the introduction of a single base change is required. The introduction of additional silent mutations designed to prevent re-cutting of the modified allele by Cas9 is not an optimal strategy, particularly for non-coding variants. Here, we developed an improved protocol for efficient engineering of single nucleotide variants in human iPS cells. Using a fluorescent BFP->GFP assay to monitor the incorporation of a single base pair change, we optimized the protocol to achieve HDR in 70% of unselected human iPS cells. The additive effects of cold shock, a small molecule enhancer of HDR and chemically modified ssODN dramatically shift the bias of repair in favor of HDR, resulting in a seven-fold higher ratio of HDR to NHEJ from 0.5 to 3.7.
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http://dx.doi.org/10.1016/j.ymeth.2019.06.016DOI Listing
July 2019

Response to "Unexpected mutations after CRISPR-Cas9 editing in vivo".

Nat Methods 2018 04 30;15(4):235-236. Epub 2018 Mar 30.

IMPC Cas9 Working Group and the IMPC Steering Committee and executive director of the IMPC (www.mousephenotype.org).

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http://dx.doi.org/10.1038/nmeth.4559DOI Listing
April 2018

Corrigendum: High-throughput discovery of novel developmental phenotypes.

Nature 2017 11 8;551(7680):398. Epub 2017 Nov 8.

This corrects the article DOI: 10.1038/nature19356.
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http://dx.doi.org/10.1038/nature24643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849394PMC
November 2017

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction.

Nat Commun 2017 10 12;8(1):886. Epub 2017 Oct 12.

Medical Research Council Harwell Institute (Mammalian Genetics Unit and Mary Lyon Centre), Harwell, Oxfordshire, OX11 0RD, UK.

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.
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http://dx.doi.org/10.1038/s41467-017-00595-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638796PMC
October 2017

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

Nucleic Acids Res 2017 Dec;45(21):e174

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.
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http://dx.doi.org/10.1093/nar/gkx811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716182PMC
December 2017

Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.

Nat Genet 2017 Aug 26;49(8):1231-1238. Epub 2017 Jun 26.

William Harvey Research Institute, Queen Mary University of London, London, UK.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.
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http://dx.doi.org/10.1038/ng.3901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546242PMC
August 2017

Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.

Sci Rep 2017 05 22;7(1):2244. Epub 2017 May 22.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.
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http://dx.doi.org/10.1038/s41598-017-01766-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440395PMC
May 2017

Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis.

Nat Commun 2017 04 25;8:15013. Epub 2017 Apr 25.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage-Chlamydia interactions in vitro. We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections.
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http://dx.doi.org/10.1038/ncomms15013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414054PMC
April 2017

One-step generation of conditional and reversible gene knockouts.

Nat Methods 2017 03 30;14(3):287-289. Epub 2017 Jan 30.

Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK.

Loss-of-function studies are key for investigating gene function, and CRISPR technology has made genome editing widely accessible in model organisms and cells. However, conditional gene inactivation in diploid cells is still difficult to achieve. Here, we present CRISPR-FLIP, a strategy that provides an efficient, rapid and scalable method for biallelic conditional gene knockouts in diploid or aneuploid cells, such as pluripotent stem cells, 3D organoids and cell lines, by co-delivery of CRISPR-Cas9 and a universal conditional intronic cassette.
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http://dx.doi.org/10.1038/nmeth.4156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777571PMC
March 2017

Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells.

Development 2017 02 17;144(4):635-648. Epub 2017 Jan 17.

MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK

Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci ( and ); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. and ); and (4) engineering of glioma mutations ( deletion; point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis.
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http://dx.doi.org/10.1242/dev.140855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312033PMC
February 2017

High-throughput discovery of novel developmental phenotypes.

Nature 2016 09 14;537(7621):508-514. Epub 2016 Sep 14.

The Jackson Laboratory, Bar Harbor, Maine 04609, USA.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
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http://dx.doi.org/10.1038/nature19356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295821PMC
September 2016

CRISPR-Cas9 enables conditional mutagenesis of challenging loci.

Sci Rep 2016 09 1;6:32326. Epub 2016 Sep 1.

Institute of Developmental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg/Munich, Germany.

The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods.
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http://dx.doi.org/10.1038/srep32326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007477PMC
September 2016

Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse.

Open Biol 2015 Dec;5(12):150209

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK

To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development.
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http://dx.doi.org/10.1098/rsob.150209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703062PMC
December 2015

Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.

Biochim Biophys Acta 2016 Jul 31;1863(7 Pt B):1728-48. Epub 2015 Oct 31.

Department of Stem Cell Biology, Centre for Biomolecular Sciences, University of Nottingham, NG7 2RD, United Kingdom.

Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.
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http://dx.doi.org/10.1016/j.bbamcr.2015.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5221745PMC
July 2016

Off-target mutations are rare in Cas9-modified mice.

Nat Methods 2015 Jun;12(6):479

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

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http://dx.doi.org/10.1038/nmeth.3408DOI Listing
June 2015

Is mouse embryonic stem cell technology obsolete?

Genome Biol 2015 May 27;16:109. Epub 2015 May 27.

Stem Cell Engineering, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Injection of recombinant Cas9 protein and synthetic guide RNAs into mouse zygotes has been shown to facilitate gene disruption and knock-ins using the CRISPR system. These technologies may soon displace genetic modification using embryonic stem cells.
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http://dx.doi.org/10.1186/s13059-015-0673-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443626PMC
May 2015

WGE: a CRISPR database for genome engineering.

Bioinformatics 2015 Sep 14;31(18):3078-80. Epub 2015 May 14.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Unlabelled: The rapid development of CRISPR-Cas9 mediated genome editing techniques has given rise to a number of online and stand-alone tools to find and score CRISPR sites for whole genomes. Here we describe the Wellcome Trust Sanger Institute Genome Editing database (WGE), which uses novel methods to compute, visualize and select optimal CRISPR sites in a genome browser environment. The WGE database currently stores single and paired CRISPR sites and pre-calculated off-target information for CRISPRs located in the mouse and human exomes. Scoring and display of off-target sites is simple, and intuitive, and filters can be applied to identify high-quality CRISPR sites rapidly. WGE also provides a tool for the design and display of gene targeting vectors in the same genome browser, along with gene models, protein translation and variation tracks. WGE is open, extensible and can be set up to compute and present CRISPR sites for any genome.

Availability And Implementation: The WGE database is freely available at www.sanger.ac.uk/htgt/wge

Contact: : vvi@sanger.ac.uk or skarnes@sanger.ac.uk

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btv308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565030PMC
September 2015

ZFP57 and the Targeted Maintenance of Postfertilization Genomic Imprints.

Cold Spring Harb Symp Quant Biol 2015 ;80:177-87

Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom.

Epigenetic modifications play an important role in modulating genome function. In mammals, inappropriate epigenetic states can cause embryonic lethality and various acquired and inherited diseases; hence, it is important to understand how such states are formed and maintained in particular genomic contexts. Genomic imprinting is a process in which epigenetic states provide a sustained memory of parental origin and cause gene expression/repression from only one of the two parental chromosomes. Genomic imprinting is therefore a valuable model to decipher the principles and processes associated with the targeting and maintenance of epigenetic states in general. Krüppel-associated box zinc finger proteins (KRAB-ZFPs) are proteins that have the potential to mediate this. ZFP57, one of the best characterized proteins in this family, has been shown to target and maintain epigenetic states at imprinting control regions after fertilization. Its role in imprinting through the use of ZFP57 mutants in mouse and the wider implications of KRAB-ZFPs for the targeted maintenance of epigenetic states are discussed here.
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http://dx.doi.org/10.1101/sqb.2015.80.027466DOI Listing
January 2018

Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects.

Nat Methods 2014 Apr 2;11(4):399-402. Epub 2014 Mar 2.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.
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http://dx.doi.org/10.1038/nmeth.2857DOI Listing
April 2014

The International Mouse Phenotyping Consortium Web Portal, a unified point of access for knockout mice and related phenotyping data.

Nucleic Acids Res 2014 Jan 4;42(Database issue):D802-9. Epub 2013 Nov 4.

European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK, Medical Research Council Harwell (Mammalian Genetics Unit and Mary Lyon Centre), Harwell, Oxfordshire OX11 0RD, UK and Mouse Informatics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

The International Mouse Phenotyping Consortium (IMPC) web portal (http://www.mousephenotype.org) provides the biomedical community with a unified point of access to mutant mice and rich collection of related emerging and existing mouse phenotype data. IMPC mouse clinics worldwide follow rigorous highly structured and standardized protocols for the experimentation, collection and dissemination of data. Dedicated 'data wranglers' work with each phenotyping center to collate data and perform quality control of data. An automated statistical analysis pipeline has been developed to identify knockout strains with a significant change in the phenotype parameters. Annotation with biomedical ontologies allows biologists and clinicians to easily find mouse strains with phenotypic traits relevant to their research. Data integration with other resources will provide insights into mammalian gene function and human disease. As phenotype data become available for every gene in the mouse, the IMPC web portal will become an invaluable tool for researchers studying the genetic contributions of genes to human diseases.
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http://dx.doi.org/10.1093/nar/gkt977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964955PMC
January 2014

Molecular characterization of mutant mouse strains generated from the EUCOMM/KOMP-CSD ES cell resource.

Mamm Genome 2013 Aug 4;24(7-8):286-94. Epub 2013 Aug 4.

The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, CB10 1SA, UK.

The Sanger Mouse Genetics Project generates knockout mice strains using the EUCOMM/KOMP-CSD embryonic stem (ES) cell collection and characterizes the consequences of the mutations using a high-throughput primary phenotyping screen. Upon achieving germline transmission, new strains are subject to a panel of quality control (QC) PCR- and qPCR-based assays to confirm the correct targeting, cassette structure, and the presence of the 3' LoxP site (required for the potential conditionality of the allele). We report that over 86 % of the 731 strains studied showed the correct targeting and cassette structure, of which 97 % retained the 3' LoxP site. We discuss the characteristics of the lines that failed QC and postulate that the majority of these may be due to mixed ES cell populations which were not detectable with the original screening techniques employed when creating the ES cell resource.
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http://dx.doi.org/10.1007/s00335-013-9467-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3745610PMC
August 2013

Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes.

Cell 2013 Jul;154(2):452-64

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP:
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http://dx.doi.org/10.1016/j.cell.2013.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717207PMC
July 2013

Accessing data from the International Mouse Phenotyping Consortium: state of the art and future plans.

Mamm Genome 2012 Oct 19;23(9-10):641-52. Epub 2012 Sep 19.

Mammalian Genetics Unit, Medical Research Council Harwell, Harwell, Oxfordshire OX11 0RD, UK.

The International Mouse Phenotyping Consortium (IMPC) (http://www.mousephenotype.org) will reveal the pleiotropic functions of every gene in the mouse genome and uncover the wider role of genetic loci within diverse biological systems. Comprehensive informatics solutions are vital to ensuring that this vast array of data is captured in a standardised manner and made accessible to the scientific community for interrogation and analysis. Here we review the existing EuroPhenome and WTSI phenotype informatics systems and the IKMC portal, and present plans for extending these systems and lessons learned to the development of a robust IMPC informatics infrastructure.
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http://dx.doi.org/10.1007/s00335-012-9428-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106044PMC
October 2012

The mammalian gene function resource: the International Knockout Mouse Consortium.

Mamm Genome 2012 Oct 12;23(9-10):580-6. Epub 2012 Sep 12.

The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1HH, UK.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
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http://dx.doi.org/10.1007/s00335-012-9422-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463800PMC
October 2012

Mouse large-scale phenotyping initiatives: overview of the European Mouse Disease Clinic (EUMODIC) and of the Wellcome Trust Sanger Institute Mouse Genetics Project.

Mamm Genome 2012 Oct 9;23(9-10):600-10. Epub 2012 Sep 9.

Institut Clinique de la Souris, PHENOMIN, IGBMC/ICS-MCI, CNRS, INSERM, Université de Strasbourg, UMR7104, UMR964, 1 rue Laurent Fries, 67404 Illkirch, France.

Two large-scale phenotyping efforts, the European Mouse Disease Clinic (EUMODIC) and the Wellcome Trust Sanger Institute Mouse Genetics Project (SANGER-MGP), started during the late 2000s with the aim to deliver a comprehensive assessment of phenotypes or to screen for robust indicators of diseases in mouse mutants. They both took advantage of available mouse mutant lines but predominantly of the embryonic stem (ES) cells resources derived from the European Conditional Mouse Mutagenesis programme (EUCOMM) and the Knockout Mouse Project (KOMP) to produce and study 799 mouse models that were systematically analysed with a comprehensive set of physiological and behavioural paradigms. They captured more than 400 variables and an additional panel of metadata describing the conditions of the tests. All the data are now available through EuroPhenome database (www.europhenome.org) and the WTSI mouse portal (http://www.sanger.ac.uk/mouseportal/), and the corresponding mouse lines are available through the European Mouse Mutant Archive (EMMA), the International Knockout Mouse Consortium (IKMC), or the Knockout Mouse Project (KOMP) Repository. Overall conclusions from both studies converged, with at least one phenotype scored in at least 80% of the mutant lines. In addition, 57% of the lines were viable, 13% subviable, 30% embryonic lethal, and 7% displayed fertility impairments. These efforts provide an important underpinning for a future global programme that will undertake the complete functional annotation of the mammalian genome in the mouse model.
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http://dx.doi.org/10.1007/s00335-012-9418-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463797PMC
October 2012

Large-scale identification of microRNA targets in murine Dgcr8-deficient embryonic stem cell lines.

PLoS One 2012 17;7(8):e41762. Epub 2012 Aug 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom.

Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3' UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041762PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422281PMC
April 2013

MiR-25 regulates Wwp2 and Fbxw7 and promotes reprogramming of mouse fibroblast cells to iPSCs.

PLoS One 2012 17;7(8):e40938. Epub 2012 Aug 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.

Background: miRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs).

Methods: We screened 52 miRNAs cloned in a piggybac (PB) vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES) cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation.

Results And Conclusion: We found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040938PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422229PMC
April 2013