Abstract
The prospect of introducing a single C-to-T change at a specific genomic location has become feasible with APOBEC-Cas9 editing technologies. We present a panel of eGFP reporters for quantification and optimization of single base editing by APOBEC-Cas9 editosomes. Reporter utility is demonstrated by comparing activities of seven human APOBEC3 enzymes and rat APOBEC1 (BE3). APOBEC3A and RNA binding-defective variants of APOBEC3B and APOBEC3H display the highest single base editing efficiencies. APOBEC3B catalytic domain complexes also elicit the lowest frequencies of adjacent off-target events. However, unbiased deep-sequencing of edited reporters shows that all editosomes have some degree of local off-target editing. Thus, further optimization is required to generate true single base editors and the eGFP reporters described here have the potential to facilitate this process.
Highlights
The single-stranded DNA cytosine to uracil (C-to-U) deamination activity of several members of the antiviral APOBEC family has been harnessed recently for site-specific genome engineering by incorporation into Cas9/ guide (g)RNA editing complexes[1,2,3,4,5,6,7,8]
As a complement to this technical necessity, we developed a mCherry restoration-of-function assay that requires APOBEC-mediated DNA editing at two adjacent sites followed by DNA breakage and double-stranded breaks (DSBs) repair by non-homologous end-joining[2]
L202 reporter sequence, and the editing event required to restore eGFP activity. (b) Representative fluorescent microscopy images of 293 T cells transfected with the L202 reporter, the APOBEC3A editosome plasmid, and a gRNA-202 or a non-specific (NS) gRNA construct. (c–f) Quantification of APOBEC editosome activities using eGFP L138 and Y93 single base editing reporters, respectively
Summary
The single-stranded DNA cytosine to uracil (C-to-U) deamination activity of several members of the antiviral APOBEC family has been harnessed recently for site-specific genome engineering by incorporation into Cas9/ guide (g)RNA editing complexes[1,2,3,4,5,6,7,8] An advantage of this technology over canonical Cas[9] editing is precise single base substitution mutations (C-to-T) without potentially detrimental intermediates and outcomes including DNA double-stranded breaks (DSBs) and insertion/deletion mutations (indels). We report the development of a panel of reporter constructs in which a single on-target C-to-T editing event restores eGFP fluorescence and enables real-time quantification of on-target DNA editing
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