Genotyping with CRISPR-Cas-derived RNA-guided endonucleases.
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.
National Creative Initiatives Research Center for Genome Engineering, Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea.
Jong Min
JM
Kim
National Creative Initiatives Research Center for Genome Engineering, Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea.
Daesik
D
Kim
ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Geumcheon-Gu, Seoul 153-023, South Korea.
Seokjoong
S
Kim
National Creative Initiatives Research Center for Genome Engineering, Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea.
Jin-Soo
JS
Kim
D001483:Base Sequence; D064113:CRISPR-Cas Systems; D004720:Endonucleases / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D012313:RNA / Q000378:metabolism*
D012313:RNA; D004720:Endonucleases