A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC-Cas9 or cleavage by Cas9 in living cells

Nucleic Acids Res. 2018 Aug 21;46(14):e84. doi: 10.1093/nar/gky332.

Abstract

Base editing is an exciting new genome engineering technology. C-to-T mutations in genomic DNA have been achieved using ribonucleoprotein complexes comprised of rat APOBEC1 single-stranded DNA deaminase, Cas9 nickase (Cas9n), uracil DNA glycosylase inhibitor (UGI), and guide (g)RNA. Here, we report the first real-time reporter system for quantification of APOBEC-mediated base editing activity in living mammalian cells. The reporter expresses eGFP constitutively as a marker for transfection or transduction, and editing restores functionality of an upstream mCherry cassette through the simultaneous processing of two gRNA binding regions that each contain an APOBEC-preferred 5'TCA target site. Using this system as both an episomal and a chromosomal editing reporter, we show that human APOBEC3A-Cas9n-UGI and APOBEC3B-Cas9n-UGI base editing complexes are more efficient than the original rat APOBEC1-Cas9n-UGI construct. We also demonstrate coincident enrichment of editing events at a heterologous chromosomal locus in reporter-edited, mCherry-positive cells. The mCherry reporter also quantifies the double-stranded DNA cleavage activity of Cas9, and may therefore be adaptable for use with many different CRISPR systems. The combination of a rapid, fluorescence-based editing reporter system and more efficient, structurally defined DNA editing enzymes broadens the versatility of the rapidly expanding toolbox of genome editing and engineering technologies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • CHO Cells
  • COS Cells
  • CRISPR-Associated Protein 9*
  • Chlorocebus aethiops
  • Cricetulus
  • Cytidine Deaminase*
  • DNA Cleavage
  • Fluorescent Dyes*
  • Gene Editing*
  • Genes, Reporter
  • Green Fluorescent Proteins*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Luminescent Proteins*
  • Minor Histocompatibility Antigens*
  • Proteins*
  • Red Fluorescent Protein

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • Minor Histocompatibility Antigens
  • Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • CRISPR-Associated Protein 9
  • APOBEC3A protein, human
  • APOBEC3B protein, human
  • Cytidine Deaminase