Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations

Nat Commun. 2014 Feb 26;5:3378. doi: 10.1038/ncomms4378.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems / genetics
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Flow Cytometry
  • Genes, Reporter / genetics
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mutation*
  • RNA, Guide / genetics
  • RNA, Guide / metabolism*
  • Sequence Homology, Nucleic Acid
  • Transfection

Substances

  • CRISPR-Associated Proteins
  • Luminescent Proteins
  • RNA, Guide
  • red fluorescent protein
  • Green Fluorescent Proteins
  • Endonucleases