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Targeted mutagenesis in a human-parasitic nematode

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      Abstract

      Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites.

      Author summary

      Parasitic worms are a widespread public health burden, yet very little is known about the cellular and molecular mechanisms that contribute to their parasitic lifestyle. One of the major barriers to better understanding these mechanisms is that there are currently no available methods for making targeted gene knockouts in any parasitic worm species. Here, we describe the first mutant phenotype in a parasitic worm resulting from a targeted gene disruption. We applied CRISPR-Cas9-mediated mutagenesis to parasitic worms in the genus Strongyloides and developed a method that overcomes many of the challenges that have previously inhibited generating mutant parasitic worms. We characterize heritable mutant phenotypes and outline a toolkit that will be applicable to many other genes with potential roles in parasitism. Importantly, we developed our method for gene knockouts in a human-parasitic worm. By directly investigating the genes and molecular pathways that enable worms to parasitize humans, we may be able to develop novel anthelmintic therapies or other measures for preventing nematode infections.

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      Most cited references 56

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      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

      In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.
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        Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data

        Summary: The two main functions of bioinformatics are the organization and analysis of biological data using computational resources. Geneious Basic has been designed to be an easy-to-use and flexible desktop software application framework for the organization and analysis of biological data, with a focus on molecular sequences and related data types. It integrates numerous industry-standard discovery analysis tools, with interactive visualizations to generate publication-ready images. One key contribution to researchers in the life sciences is the Geneious public application programming interface (API) that affords the ability to leverage the existing framework of the Geneious Basic software platform for virtually unlimited extension and customization. The result is an increase in the speed and quality of development of computation tools for the life sciences, due to the functionality and graphical user interface available to the developer through the public API. Geneious Basic represents an ideal platform for the bioinformatics community to leverage existing components and to integrate their own specific requirements for the discovery, analysis and visualization of biological data. Availability and implementation: Binaries and public API freely available for download at http://www.geneious.com/basic, implemented in Java and supported on Linux, Apple OSX and MS Windows. The software is also available from the Bio-Linux package repository at http://nebc.nerc.ac.uk/news/geneiousonbl. Contact: peter@biomatters.com
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          The genetics of Caenorhabditis elegans.

          Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
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            Author and article information

            Affiliations
            [1 ] Molecular Biology Institute, University of California, Los Angeles, California, United States of America
            [2 ] Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California, United States of America
            [3 ] Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, California, United States of America
            [4 ] Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California, United States of America
            George Washington University School of Medicine and Health Sciences, UNITED STATES
            Author notes

            The authors have declared that no competing interests exist.

            Contributors
            Role: Conceptualization, Role: Formal analysis, Role: Investigation, Role: Methodology, Role: Writing – original draft, Role: Writing – review & editing
            Role: Conceptualization, Role: Formal analysis, Role: Investigation, Role: Methodology, Role: Writing – review & editing
            ORCID: http://orcid.org/0000-0002-0887-2044, Role: Formal analysis, Role: Investigation, Role: Writing – review & editing
            ORCID: http://orcid.org/0000-0001-8187-4292, Role: Investigation
            ORCID: http://orcid.org/0000-0002-9352-5927, Role: Formal analysis
            ORCID: http://orcid.org/0000-0002-1765-8103, Role: Investigation
            ORCID: http://orcid.org/0000-0001-9355-9564, Role: Supervision
            ORCID: http://orcid.org/0000-0003-0260-3174, Role: Conceptualization, Role: Formal analysis, Role: Funding acquisition, Role: Project administration, Role: Supervision, Role: Writing – original draft, Role: Writing – review & editing
            Role: Editor
            Journal
            PLoS Pathog
            PLoS Pathog
            plos
            plospath
            PLoS Pathogens
            Public Library of Science (San Francisco, CA USA )
            1553-7366
            1553-7374
            10 October 2017
            October 2017
            : 13
            : 10
            29016680 5650185 10.1371/journal.ppat.1006675 PPATHOGENS-D-17-01106
            © 2017 Gang et al

            This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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            Figures: 6, Tables: 0, Pages: 31
            Product
            Funding
            Funded by: funder-id http://dx.doi.org/10.13039/100000861, Burroughs Wellcome Fund;
            Award Recipient : ORCID: http://orcid.org/0000-0003-0260-3174
            Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
            Award ID: 1DP2DC014596-01
            Award Recipient : ORCID: http://orcid.org/0000-0003-0260-3174
            Funded by: funder-id http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
            Award Recipient : ORCID: http://orcid.org/0000-0003-0260-3174
            This work was funded by the Whitcome Predoctoral Training Program, UCLA Molecular Biology Institute, Ruth L. Kirschstein National Research Service Award AI007323, and National Science Foundation East Asia and Pacific Summer Institute Fellowship Award 1414655 (SSG); the UCLA Undergraduate Research Scholar Program (EY); the UCLA-Howard Hughes Medical Institute Pathways to Success Program (JBL); and a Burroughs-Wellcome Fund Investigators in the Pathogenesis of Disease Award, NIH New Innovator Award 1DP2DC014596, and Howard Hughes Medical Institute Faculty Scholar Award (EAH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology and Life Sciences
            Organisms
            Eukaryota
            Animals
            Invertebrates
            Nematoda
            Strongyloides
            Strongyloides Stercoralis
            Medicine and Health Sciences
            Parasitic Diseases
            Nematode Infections
            Biology and Life Sciences
            Genetics
            Mutagenesis
            Research and Analysis Methods
            Experimental Organism Systems
            Model Organisms
            Caenorhabditis Elegans
            Research and Analysis Methods
            Model Organisms
            Caenorhabditis Elegans
            Research and Analysis Methods
            Experimental Organism Systems
            Animal Models
            Caenorhabditis Elegans
            Biology and Life Sciences
            Organisms
            Eukaryota
            Animals
            Invertebrates
            Nematoda
            Caenorhabditis
            Caenorhabditis Elegans
            Biology and Life Sciences
            Biotechnology
            Bioengineering
            Synthetic Bioengineering
            Genome Engineering
            Synthetic Genome Editing
            Crispr
            Engineering and Technology
            Bioengineering
            Synthetic Bioengineering
            Genome Engineering
            Synthetic Genome Editing
            Crispr
            Biology and Life Sciences
            Synthetic Biology
            Synthetic Bioengineering
            Genome Engineering
            Synthetic Genome Editing
            Crispr
            Engineering and Technology
            Synthetic Biology
            Synthetic Bioengineering
            Genome Engineering
            Synthetic Genome Editing
            Crispr
            Biology and Life Sciences
            Synthetic Biology
            Synthetic Genomics
            Synthetic Genome Editing
            Crispr
            Engineering and Technology
            Synthetic Biology
            Synthetic Genomics
            Synthetic Genome Editing
            Crispr
            Biology and Life Sciences
            Biotechnology
            Genetic Engineering
            Plasmid Vectors
            Biology and Life Sciences
            Biochemistry
            Proteins
            Ribonucleoproteins
            Biology and Life Sciences
            Molecular Biology
            Molecular Biology Techniques
            Artificial Gene Amplification and Extension
            Polymerase Chain Reaction
            Research and Analysis Methods
            Molecular Biology Techniques
            Artificial Gene Amplification and Extension
            Polymerase Chain Reaction
            Custom metadata
            vor-update-to-uncorrected-proof
            2017-10-20
            All relevant data are within the paper and its Supporting Information files.

            Infectious disease & Microbiology

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