Browse

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

Cited 118 time in Web of Science Cited 74 time in Scopus
Authors
Kim, Jong Min; Kim, Daesik; Kim, Seokjoong; Kim, Jin-Soo
Issue Date
2014-01
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
http://hdl.handle.net/10371/165641
DOI
https://doi.org/10.1038/ncomms4157
Files in This Item:
Appears in Collections:
Seoul National University(서울대학교)Featured Researcher's Articles
  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse