Improved genome editing in human cell lines using the CRISPR method.
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 mutatio...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0109752 |
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| _version_ | 1850161602918088704 |
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| author | Ivan M Munoz Piotr Szyniarowski Rachel Toth John Rouse Christophe Lachaud |
| author_facet | Ivan M Munoz Piotr Szyniarowski Rachel Toth John Rouse Christophe Lachaud |
| author_sort | Ivan M Munoz |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-2491b77152034ba5aaaedd6f6c00bc82 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-2491b77152034ba5aaaedd6f6c00bc822025-08-20T02:22:46ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-01910e10975210.1371/journal.pone.0109752Improved genome editing in human cell lines using the CRISPR method.Ivan M MunozPiotr SzyniarowskiRachel TothJohn RouseChristophe LachaudThe 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.https://doi.org/10.1371/journal.pone.0109752 |
| spellingShingle | Ivan M Munoz Piotr Szyniarowski Rachel Toth John Rouse Christophe Lachaud Improved genome editing in human cell lines using the CRISPR method. PLoS ONE |
| title | Improved genome editing in human cell lines using the CRISPR method. |
| title_full | Improved genome editing in human cell lines using the CRISPR method. |
| title_fullStr | Improved genome editing in human cell lines using the CRISPR method. |
| title_full_unstemmed | Improved genome editing in human cell lines using the CRISPR method. |
| title_short | Improved genome editing in human cell lines using the CRISPR method. |
| title_sort | improved genome editing in human cell lines using the crispr method |
| url | https://doi.org/10.1371/journal.pone.0109752 |
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