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  • 1
    Online Resource
    Online Resource
    Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes
    Wiley ; 2020
    In:  Current Protocols in Molecular Biology Vol. 130, No. 1 ( 2020-03)
    Details
    In: Current Protocols in Molecular Biology, Wiley, Vol. 130, No. 1 ( 2020-03)
    Abstract: The CRISPR‐Cas9 system makes it possible to cause double‐strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or ‘knock‐in’ of an exogenous cassette. One common application of knock‐in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock‐in of fluorescent protein ORFs into Cas9‐expressing Drosophila S2R+ cultured cells, the single‐stranded DNA (ssDNA) Drop‐In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop‐In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1 : Knock‐in into Cas9‐positive S2R+ cells using the ssDNA Drop‐In approach Basic Protocol 2 : Knock‐in into Cas9‐positive S2R+ cells by homology‐independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1 : sgRNA design and cloning Support Protocol 2 : ssDNA donor synthesis Support Protocol 3 : Transfection using Effectene Support Protocol 4 : Electroporation of S2R+‐MT::Cas9 Drosophila cells Support Protocol 5 : Single‐cell isolation of fluorescent cells using FACS
    Type of Medium: Online Resource
    ISSN: 1934-3639 , 1934-3647
    URL: Issue
    URL: Article
    DOI: 10.1002/cpmb.v130.1
    DOI: 10.1002/cpmb.112
    Language: English
    Publisher: Wiley
    Publication Date: 2020
    detail.hit.zdb_id: 2179066-8
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  • 2
    Online Resource
    Online Resource
    Protein Knockouts in Living Eukaryotes Using deGradFP and Green Fluorescent Protein Fusion Targets
    Wiley ; 2013
    In:  Current Protocols in Protein Science Vol. 73, No. 1 ( 2013-08)
    Details
    In: Current Protocols in Protein Science, Wiley, Vol. 73, No. 1 ( 2013-08)
    Abstract: This unit describes deGradFP (degrade Green Fluorescent Protein), an easy‐to‐implement protein knockout method applicable in any eukaryotic genetic system. Depleting a protein in order to study its function in a living organism is usually achieved at the gene level (genetic mutations) or at the RNA level (RNA interference and morpholinos). However, any system that acts upstream of the proteic level depends on the turnover rate of the existing target protein, which can be extremely slow. In contrast, deGradFP is a fast method that directly depletes GFP fusion proteins. In particular, deGradFP is able to counteract maternal effects in embryos and causes early and fast onset loss‐of‐function phenotypes of maternally contributed proteins. Curr. Protoc. Protein Sci . 73:30.2.1‐30.2.13. ‐ 2013 by John Wiley & Sons, Inc.
    Type of Medium: Online Resource
    ISSN: 1934-3655 , 1934-3663
    URL: Issue
    URL: Article
    DOI: 10.1002/0471140864.2013.73.issue-1
    DOI: 10.1002/0471140864.ps3002s73
    Language: English
    Publisher: Wiley
    Publication Date: 2013
    detail.hit.zdb_id: 2179077-2
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