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Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

Cited 133 time in Web of Science Cited 110 time in Scopus
Authors

Kim, Jong Min; Kim, Daesik; Kim, Seokjoong; Kim, Jin-Soo

Issue Date
2014-01
Publisher
Nature Publishing Group
Citation
Nature Communications, Vol.5, p. 3157
Abstract
Restriction fragment length polymorphism (RFLP) analysis is one of the oldest, most convenient and least expensive methods of genotyping, but is limited by the availability of restriction endonuclease sites. Here we present a novel method of employing CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) in RFLP analysis. We prepare RGENs by complexing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed guide RNAs that are complementary to the DNA sequences of interest. Then, we genotype recurrent mutations found in cancer and small insertions or deletions (indels) induced in cultured cells and animals by RGENs and other engineered nucleases such as transcription activator-like effector nucleases (TALENs). Unlike T7 endonuclease I or Surveyor assays that are widely used for genotyping engineered nuclease-induced mutations, RGEN-mediated RFLP analysis can detect homozygous mutant clones that contain identical biallelic indel sequences and is not limited by sequence polymorphisms near the nuclease target sites.
ISSN
2041-1723
URI
https://hdl.handle.net/10371/165641
DOI
https://doi.org/10.1038/ncomms4157
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  • College of Natural Sciences
  • Department of Chemistry
Research Area Biology and Biochemistry

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