EditR: A Method to Quantify Base Editing from Sanger Sequencing

TLDR

Base editors—CRISPR‑Cas9‑cytidine deaminase fusions—enable precise C:G→T:A conversion without double‑strand breaks, offering unprecedented efficiency but current outcome analyses are imprecise or costly. We developed EditR, a free, cost‑effective program that quantifies base‑editing efficiency from a single Sanger sequencing file and guide‑RNA sequence. EditR analyzes the Sanger trace to calculate editing percentages at each position and is adaptable to adenosine deaminase editors that convert A:T→G:C. EditR outperforms enzymatic assays, delivers detailed positional and efficiency insights, and provides a robust, inexpensive tool that broadens base‑editing applications and drives further innovation.

Abstract

CRISPR-Cas9-Cytidine deaminase fusion enzymes—termed “base editors”—allow targeted editing of genomic deoxycytidine to deoxythymidine (C:G→T:A) without the need for double-stranded break induction. Base editors represent a paradigm shift in gene editing technology due to their unprecedented efficiency to mediate targeted, single-base conversion. However, current analysis of base editing outcomes rely on methods that are either imprecise or expensive and time-consuming. To overcome these limitations, we developed a simple, cost-effective, and accurate program to measure base editing efficiency from fluorescence-based Sanger sequencing, termed “EditR.” We provide EditR as a free online tool or downloadable desktop application requiring a single Sanger sequencing file and guide RNA sequence. EditR is more accurate than enzymatic assays, and provides added insight to the position, type, and efficiency of base editing. Furthermore, EditR is likely amenable to quantify base editing from the recently developed adenosine deaminase base editors that act on either DNA (adenosine deaminase base editors [ABEs]) or RNA (REPAIRs) (catalyzes A:T→G:C). Collectively, we demonstrate that EditR is a robust, inexpensive tool that will facilitate the broad application of base editing technology, thereby fostering further innovation in this burgeoning field.