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

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.

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).

Figure 2.

High-efficiency editing by human A3A and A3Bctd editosomes. (A) Representative fluorescence microscopy images of ACE-activated, mCherry-positive 293T cells catalyzed by human A3A, human A3Bctd, or rat APOBEC1/BE3 editosomes (mCherry codon 59-directed gRNA versus NS-gRNA; inset white bar = 30 μm). (B) Quantification of the experiment in panel ‘A’ together with 2 independent parallel experiments (n = 3; average ± SD). The corresponding immunoblots of expressed APOBEC–Cas9n-UGI constructs are shown below (low and high exposures to help visualize BE3) with HSP90 as a loading control. (C) Time course of ACE activation in 293T cells catalyzed by human A3A, human A3Bctd, or rat APOBEC1/BE3 editosomes (mCherry codon 59-directed gRNA versus NS-gRNA; n = 3; mean ± SD; error bars smaller than symbols are not shown). (D) Titration data for 293T cells co-transfected with the ACE reporter, mCherry codon 59-directed gRNA, and different amounts of the indicated editosome constructs (100–600 ng; n = 3; mean ± SD). BE3i has an intron in the rat APOBEC1 portion of the construct, identical to the intron required for propagation of A3A and A3Bctd constructs in E. coli and for expression in mammalian cells.

Figure 3.

Chromosomal editing by A3A and A3Bctd editosomes. (A) Editing of single copy genomic ACE reporter by the indicated editosomes in 293T and HeLa cells (n = 3, average ± SD). (B) Immunoblots corresponding to a representative experiment in panel A showing APOBEC–Cas9n-UGI expression levels and HSP90 as a loading control. (C) Sanger sequencing results for the gRNA-binding region of the ACE reporter recovered by high-fidelity PCR of mCherry-positive 293T. Mutated nucleotides are depicted in red and deleted nucleotides by hyphens. The number of times each sequence was recovered is indicated to the right.

Figure 4.

ACE reporter activation through Cas9 nucleolytic cleavage. (A) Quantification of ACE reporter activation in 293T cells 72 h after co-transfection of ACE reporter, mCherry codon 59 targeting gRNA or NS-gRNA, and A3A-Cas9n-UGI, A3Bctd-Cas9n-UGI, rat APOBEC1-Cas9n-UGI/BE3, Cas9, or Cas9n expression constructs (n = 3; mean ± SD). (B) Anti-Cas9 and anti-HSP90 immunoblots from a representative experiment reported in panel A.

Figure 5.

ACE enriches for base-editing events at heterologous genomic loci. (A) Schematic of a co-transfection experiment resulting in ACE reporter activation (yellow shading represents mCherry and eGFP double-positive cells). FANCF and the PstI restriction assay used to quantify chromosomal base editing of this locus. Base editing events destroy the PstI cleavage site and block cleavage of the 452 bp amplicon into 260 and 192 bp products. (B) Representative agarose gels images showing the results of FANCF base editing by A3A and A3Bctd editsomes in 293T cells. The percentage of base editing was calculated by dividing the percentage of substrate band by the total of substrate and product bands following PstI cleavage for both unsorted and mCherry-positive cell populations. (C) Sanger sequencing results for the gRNA-binding region of the FANCF gene, which was recovered by high-fidelity PCR using genomic DNA from mCherry-positive 293T. Mutated nucleotides are highlighted in red and deleted nucleotides are indicated by hyphens. The number of times each sequence was recovered is indicated to the right.