Skip to main content
. 2018 May 9;46(14):e84. doi: 10.1093/nar/gky332

Figure 1.

Figure 1.

A real-time fluorescent reporter for APOBEC- and Cas9-mediated editing. (A) Schematic of the APOBEC- and Cas9-mediated editing (ACE) reporter in the context of a lentiviral construct with a CMV promoter that drives expression of a bicistronic message encoding mutant mCherry and wild-type eGFP. The sequence of the gRNA displaced DNA strand is shown below with flanking APOBEC 5′-TCA deamination hotspots (red), PAM sites, and 43 bp insertion labeled. See text for a description of editing, cleavage, and processing events required for reversion to mCherry-positive. Ribbon schematics of defective mCherry (gray) and functionally restored mCherry (red) with the flexible loop position of residue 59 shown (model based on pdb 2H5Q). (B) Schematic of an APOBEC–Cas9n/gRNA editosome engaging a DNA target. C-to-U editing occurs in the ssDNA loop displaced by gRNA annealing to target DNA. (C) Representative images of mCherry-positive 293T cells catalyzed by BE3 and mCherry codon 59-directed gRNA (#59-gRNA) but not with NS-gRNA (NS, non-specific; inset white bar = 30 μm). (D) Quantification of the base editing experiment in panel C (n = 3; average ± SD).