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.

Single‐nucleotide editing: From principle, optimization to application

“…It has been noted that human APOBEC3A‐cas9 fusion can effectively induce efficient base editing in both methylated DNA regions and GpC dinucleotides, thus expanding the scope of base editing (X. Wang et al, ; Zong et al, ). In addition, human APOBEC3A‐Cas9n‐UGI and APOBEC3B‐Cas9n‐UGI base‐editing complexes are more efficient than the original rat APOBEC1‐Cas9n‐UGI construct (St Martin et al, ).Cas9 high‐fidelity variant (Cas9‐HF), which contains specific point mutations, is thought to have less binding energy with DNA than wild type Cas9 (wtCas9). The mutations presumably disrupt hydrogen bonding with the phosphate backbone of the complementary DNA strand, thereby decreasing Cas9 binding with mismatched sequences and increasing its overall specificity (Kleinstiver et al, ; Kleinstiver, Prew, Tsai, Topkar et al, ; Liang, Sun et al, ).…”

“…It has been noted that human APOBEC3A‐cas9 fusion can effectively induce efficient base editing in both methylated DNA regions and GpC dinucleotides, thus expanding the scope of base editing (X. Wang et al, ; Zong et al, ). In addition, human APOBEC3A‐Cas9n‐UGI and APOBEC3B‐Cas9n‐UGI base‐editing complexes are more efficient than the original rat APOBEC1‐Cas9n‐UGI construct (St Martin et al, ).…”

The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses

“…The CRISPR/Cas9 system consists of two core elements, a Cas9 endonuclease and a gRNA linked to a scaffolding tracrRNA. 11 The 20 to 25 nucleotides long gRNA targeting specific sequences can be designed synthetically and cloned into various expression vectors having Cas9 protein and tracrRNA. 12 These vectors ensure the accurate delivery of gRNA-Cas9 complex onto the specific target sequence.…”

“…19 This "base editing system" work by coupling a Cas9 nickase with enzymatic chemicals or catalysts, for example, cytidine deaminase that causes direct genetic manipulations by converting one base to another (C→T) through inhibition of base excision repair system. 20 Another base editor system dCas9-AIDx can change cytidine to other three alternative bases, thus generating a diversity of variants at desired loci. 21 Altogether, these modifications in Cas9 system have extended its application in many folds.…”

“…Very recently, a new approach of CRISPR/Cas9 has been developed which induces point mutations in the target region of DNA without requiring any single or dsDNA break and a donor template relative to HDR thus decreasing the level of InDels . This “base editing system” work by coupling a Cas9 nickase with enzymatic chemicals or catalysts, for example, cytidine deaminase that causes direct genetic manipulations by converting one base to another (C→T) through inhibition of base excision repair system . Another base editor system dCas9‐AIDx can change cytidine to other three alternative bases, thus generating a diversity of variants at desired loci .…”