Improved genome editing in human cell lines using the CRISPR method

Ivan M. Munoz, Piotr Szyniarowski, Rachel Toth, John Rouse (Lead / Corresponding author), Christophe Lachaud (Lead / Corresponding author)

    Research output: Contribution to journalArticle

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    Abstract

    The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.
    Original languageEnglish
    Article numbere109752
    Number of pages6
    JournalPLoS ONE
    Volume9
    Issue number10
    DOIs
    Publication statusPublished - 10 Oct 2014

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    Clustered Regularly Interspaced Short Palindromic Repeats
    human cell lines
    gene targeting
    Genes
    Cells
    Cell Line
    genome
    nucleases
    DNA repair
    hypersensitivity
    Guide RNA
    genes
    methodology
    cell lines
    RNA
    mutation
    genomics
    Mutagens
    phenotype
    loci

    Cite this

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    abstract = "The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29{\%}, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.",
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    Improved genome editing in human cell lines using the CRISPR method. / Munoz, Ivan M.; Szyniarowski, Piotr; Toth, Rachel; Rouse, John (Lead / Corresponding author); Lachaud, Christophe (Lead / Corresponding author).

    In: PLoS ONE, Vol. 9, No. 10, e109752, 10.10.2014.

    Research output: Contribution to journalArticle

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    AU - Toth, Rachel

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    AU - Lachaud, Christophe

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    AB - The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.

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