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

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

Author information

1
Department of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, Center for Genome Engineering, Institute for Molecular Virology, University of Minnesota, MN 55455, USA.
2
Istituto Toscano Tumori, Firenze 50134, Italy.
3
Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA.

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.

PMID:
29746667
PMCID:
PMC6101615
DOI:
10.1093/nar/gky332
[Indexed for MEDLINE]
Free PMC Article

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