• Login
    View Item 
    •   Home
    • UMass Chan Student Research and Publications
    • Morningside Graduate School of Biomedical Sciences
    • Morningside Graduate School of Biomedical Sciences Scholarly Publications
    • View Item
    •   Home
    • UMass Chan Student Research and Publications
    • Morningside Graduate School of Biomedical Sciences
    • Morningside Graduate School of Biomedical Sciences Scholarly Publications
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of eScholarship@UMassChanCommunitiesPublication DateAuthorsUMass Chan AffiliationsTitlesDocument TypesKeywordsThis CollectionPublication DateAuthorsUMass Chan AffiliationsTitlesDocument TypesKeywords

    My Account

    LoginRegister

    Help

    AboutSubmission GuidelinesData Deposit PolicySearchingAccessibilityTerms of UseWebsite Migration FAQ

    Statistics

    Most Popular ItemsStatistics by CountryMost Popular Authors

    A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Thumbnail
    Name:
    Mello_1069.full.pdf
    Size:
    3.219Mb
    Format:
    PDF
    Download
    Authors
    Kim, Heesun
    Ishidate, Takao
    Ghanta, Krishna S.
    Seth, Meetu
    Conte, Darryl Jr.
    Shirayama, Masaki
    Mello, Craig C.
    Student Authors
    Heesun Kim; Krishna S. Ghanta
    UMass Chan Affiliations
    RNA Therapeutics Institute
    Program in Molecular Medicine
    Document Type
    Journal Article
    Publication Date
    2014-08-01
    Keywords
    Animals; Base Sequence; CRISPR-Associated Proteins; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Deoxyribonucleases; Genetic Markers; *Genome, Helminth; Homologous Recombination; Molecular Sequence Data; Mutagenesis, Insertional; Plasmids; Sequence Analysis, DNA
    CRISPR-Cas9 system
    Co-CRISPR
    CRISPR-Cas9-mediated HR
    CRISPR-Cas9-induced indels
    blasticidin selection
    Computational Biology
    Genomics
    Molecular Biology
    Molecular Genetics
    
    Metadata
    Show full item record
    Link to Full Text
    http://dx.doi.org/10.1534/genetics.114.166389
    Abstract
    Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.
    Source
    Genetics. 2014 Aug;197(4):1069-80. doi: 10.1534/genetics.114.166389. Epub 2014 May 30. Link to article on publisher's site
    DOI
    10.1534/genetics.114.166389
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/33369
    PubMed ID
    24879462
    Related Resources
    Link to Article in PubMed
    Rights

    Copyright © 2014 by the Genetics Society of America. Available freely online through the author-supported open access option.

    ae974a485f413a2113503eed53cd6c53
    10.1534/genetics.114.166389
    Scopus Count
    Collections
    Morningside Graduate School of Biomedical Sciences Scholarly Publications

    entitlement

     

    Related items

    Showing items related by title, author, creator and subject.

    • Thumbnail

      PIE-1, SUMOylation, and Epigenetic Regulation of Germline Specification in Caenorhabditis elegans

      Kim, Heesun (2018-07-10)
      In many organisms, the most fundamental event during embryogenesis is differentiating between germline cells and specialized somatic cells. In C. elegans, PIE-1 functions to protect the germline from somatic differentiation and appears to do so by blocking transcription and by preventing chromatin remodeling in the germline during early embryogenesis. Yet the molecular mechanisms by which PIE-1 specifies germline remain poorly understood. Our work shows that SUMOylation facilitates PIE-1-dependent germline maintenance and specification. In vivo SUMO purification in various CRISPR strains revealed that PIE-1 is SUMOylated at lysine 68 in the germline and that this SUMOylation is essential for forming NuRD complex and preserving HDA-1 activity. Moreover, HDA-1 SUMOylation is dependent on PIE-1 and enhanced by PIE-1 SUMOylation, which is required for protecting germline integrity. Our results suggest the importance of SUMOylation in the germline maintenance and exemplify simultaneous SUMOylation of proteins in the same functional pathway.
    • Thumbnail

      Functional Genomics of Mammalian Innate Immunity

      Kiritsy, Michael C. (2020-08-31)
      The breadth of genetic diversity in the mammalian immune response stands out amongst the ubiquity of variation seen in the genome, evidence that microbial infections have been a major driver of evolution. As technology has facilitated an understanding of the etiology of immunological diversity, so too has it enabled the assessment of its varied functions. Functional genomics, with its ability to assess both cause and effect, has revolutionized our understanding of fundamental biological phenomena and recalibrated our hypotheses. We build upon the model of host immunity established by rare genetic variants that are causative of immunodeficiencies, but that incompletely consider the complexities of the genome. To expand our understanding, we performed a series of forward genetic screens to identify regulators of distinct functions of the innate immune system. Our studies discovered genes with novel functions in antigen presentation and immunoregulation, including several involved in central metabolism. Studies in macrophages and dendritic cells identified mitochondrial respiration as a positive regulator of the interferon-gamma response, and cells incapable of respiration failed to activate T cells. Notably, human mutations in several of these genes are responsible for immune dysfunction. In summary, this work uses new methods in genetic engineering to systematically assess the regulation of innate immunity. Our results suggest that variation in these regulatory pathways is likely to alter immunity in states of health and disease. Thus, our work validates a new approach to identify candidate genes relevant to immune dysfunction.
    • Thumbnail

      Anti-CRISPR Proteins: Applications in Genome Engineering

      Lee, Jooyoung (2020-07-14)
      Clustered, regularly interspaced, short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) constitute a bacterial and archaeal adaptive immune system. The ongoing arms race between prokaryotic hosts and their invaders such as phages led to the emergence of anti-CRISPR proteins as countermeasures against the potent antiviral defense. Since the first examples of anti-CRISPRs were shown in a subset of CRISPR-Cas systems, we endeavored to uncover these naturally-occurring inhibitors that inactivate different types of CRISPR-Cas systems. In the first part of my thesis, we have identified and characterized Type II anti-CRISPR proteins that inactivate several Cas9 orthologs. We share mechanistic insights into anti-CRISPR inhibition and show evidence of its potential utility as an off-switch for Cas9-mediated mammalian genome editing. Although the RNA programmability of Cas9 enables facile genetic manipulation with great potential for biotechnology and therapeutics, limitations and safety issues remain. The advent of anti-CRISPR proteins presents opportunities to exploit the inhibitors to exert temporal, conditional, or spatial control over CRISPR. In the second part of my thesis, we demonstrate that anti-CRISPR proteins can serve as useful tools for Cas9 genome editing. In particular, we have demonstrated that anti-CRISPRs are effective as genome editing off-switches in the tissues of adult mammals, and we further engineered anti-CRISPR proteins to achieve tissue-specific editing in vivo. Taken together, my thesis research aimed to mine for natural anti-CRISPR protein inhibitors and repurpose these proteins to complement current Cas9 technologies in basic and clinical research.
    DSpace software (copyright © 2002 - 2023)  DuraSpace
    Lamar Soutter Library, UMass Chan Medical School | 55 Lake Avenue North | Worcester, MA 01655 USA
    Quick Guide | escholarship@umassmed.edu
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.