Detection of genome-edited mutant clones by a simple competition-based PCR method.
Genome editing by the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) system is a revolutionary strategy to study gene functions. Since the efficiency of gene disruption in cell culture does not reach 100% typically, cloning of mutant cells is of...
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5460891?pdf=render |
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doaj-78c89f6dd2dc4cdc864a5e93f11c9e0b2020-11-24T20:41:38ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01126e017916510.1371/journal.pone.0179165Detection of genome-edited mutant clones by a simple competition-based PCR method.Takeshi HarayamaHoward RiezmanGenome editing by the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) system is a revolutionary strategy to study gene functions. Since the efficiency of gene disruption in cell culture does not reach 100% typically, cloning of mutant cells is often performed to obtain fully mutated cells. Therefore, a method to discriminate accurately mutated clones easily and quickly is crucial to accelerate the research using CRISPR/Cas9. Here, we show that knockout cells can be discriminated by a competition-based PCR, using a mixture of three primers, among which one primer overlaps with the Cas9 cleavage site. Together, we show how to optimize primer design in order to improve the effectiveness of the discrimination. Finally, we applied this method to show that mutations conferring drug resistance can be detected with high accuracy. The provided method is easy to perform and requires only basic laboratory equipment, making it suitable for almost all laboratories.http://europepmc.org/articles/PMC5460891?pdf=render |
| collection |
DOAJ |
| language |
English |
| format |
Article |
| sources |
DOAJ |
| author |
Takeshi Harayama Howard Riezman |
| spellingShingle |
Takeshi Harayama Howard Riezman Detection of genome-edited mutant clones by a simple competition-based PCR method. PLoS ONE |
| author_facet |
Takeshi Harayama Howard Riezman |
| author_sort |
Takeshi Harayama |
| title |
Detection of genome-edited mutant clones by a simple competition-based PCR method. |
| title_short |
Detection of genome-edited mutant clones by a simple competition-based PCR method. |
| title_full |
Detection of genome-edited mutant clones by a simple competition-based PCR method. |
| title_fullStr |
Detection of genome-edited mutant clones by a simple competition-based PCR method. |
| title_full_unstemmed |
Detection of genome-edited mutant clones by a simple competition-based PCR method. |
| title_sort |
detection of genome-edited mutant clones by a simple competition-based pcr method. |
| publisher |
Public Library of Science (PLoS) |
| series |
PLoS ONE |
| issn |
1932-6203 |
| publishDate |
2017-01-01 |
| description |
Genome editing by the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) system is a revolutionary strategy to study gene functions. Since the efficiency of gene disruption in cell culture does not reach 100% typically, cloning of mutant cells is often performed to obtain fully mutated cells. Therefore, a method to discriminate accurately mutated clones easily and quickly is crucial to accelerate the research using CRISPR/Cas9. Here, we show that knockout cells can be discriminated by a competition-based PCR, using a mixture of three primers, among which one primer overlaps with the Cas9 cleavage site. Together, we show how to optimize primer design in order to improve the effectiveness of the discrimination. Finally, we applied this method to show that mutations conferring drug resistance can be detected with high accuracy. The provided method is easy to perform and requires only basic laboratory equipment, making it suitable for almost all laboratories. |
| url |
http://europepmc.org/articles/PMC5460891?pdf=render |
| work_keys_str_mv |
AT takeshiharayama detectionofgenomeeditedmutantclonesbyasimplecompetitionbasedpcrmethod AT howardriezman detectionofgenomeeditedmutantclonesbyasimplecompetitionbasedpcrmethod |
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