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Cho, Seung Woo
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Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations

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Title
Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations
Author
Ramakrishna, SureshCho, Seung WooKim, SojungSong, MyungjaeGopalappa, RamuKim, Jin-SooKim, Hyongbum
Issue Date
2014-02
Publisher
NATURE PUBLISHING GROUP
Citation
NATURE COMMUNICATIONS, v.5, pp.3378
Abstract
RNA-guided endonucleases (RGENs), which are based on the clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system, have recently emerged as a simple and efficient tool for genome editing. However, the activities of prepared RGENs are sometimes low, hampering the generation of cells containing RGEN-induced mutations. Here we report efficient methods to enrich cells containing RGEN-induced mutations by using surrogate reporters. HEK293T cells are cotransfected with the reporter plasmid, a plasmid encoding Cas9 and a plasmid encoding crRNA and tracrRNA, and subjected to flow cytometric sorting, magnetic separation or hygromycin selection. The selected cell populations are highly enriched with cells containing RGEN-induced mutations, by a factor of up to 11-fold as compared with the unselected population. The fold enrichment tends to be high when RGEN activity is low. We envision that these reporters will facilitate the use of RGEN in a wide range of biomedical research.
URI
https://scholarworks.unist.ac.kr/handle/201301/25767
URL
https://www.nature.com/articles/ncomms4378
DOI
10.1038/ncomms4378
ISSN
2041-1723
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BME_Journal Papers
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