A generic strategy for CRISPR-Cas9-mediated gene tagging

Nat Commun. 2015 Dec 17;6:10237. doi: 10.1038/ncomms10237.

Abstract

Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease that can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, the introduction of foreign DNA sequences to tag an endogenous gene is still cumbersome as it requires the synthesis or cloning of homology templates. Here we present a strategy that enables the tagging of endogenous loci using one generic donor plasmid. It contains the tag of interest flanked by two gRNA recognition sites that allow excision of the tag from the plasmid. Co-transfection of cells with Cas9, a gRNA specifying the genomic locus of interest, the donor plasmid and a cassette-specific gRNA triggers the insertion of the tag by a homology-independent mechanism. The strategy is efficient and delivers clones that display a predictable integration pattern. As showcases we generated NanoLuc luciferase- and TurboGFP-tagged reporter cell lines.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • DNA / genetics*
  • Deoxyribonuclease I
  • Endonucleases
  • Genes, Reporter / genetics
  • Genetic Engineering / methods*
  • Genome, Human / genetics*
  • Green Fluorescent Proteins / genetics
  • Humans
  • Luciferases / genetics
  • Microscopy, Fluorescence
  • Plasmids
  • RNA, Guide / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Bacterial Proteins
  • RNA, Guide
  • Green Fluorescent Proteins
  • DNA
  • Luciferases
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases
  • Deoxyribonuclease I