MultiEditR: An easy validation method for detecting and quantifying RNA editing from Sanger sequencing

Abstract

RNA editing is the base change that results from RNA deamination by two predominant classes of deaminases; the APOBEC family and the ADAR family. Respectively, deamination of nucleobases by these enzymes are responsible for endogenous editing of cytosine to uracil (Cto-U) and adenosine to inosine (A-to-I). RNA editing is known to play an essential role both in maintaining normal cellular function, as well as altered cellular physiology during oncogenesis and tumour progression. Analysis of RNA editing in these important processes, largely relies on RNA-seq technology for the detection and quantification of RNA editing sites. Despite the power of these technologies, multiple sources of error in detecting and measuring base editing still exist, therefore additional validation and quantification of editing through Sanger sequencing is still required for confirmation of editing. Depending on the number of RNA editing sites that are of interest, this validation step can be both expensive and time-consuming. To address this need we developed the tool MultiEditR which provides a simple, and cost-effective method of detecting and quantifying RNA editing form Sanger sequencing. We expect that MultiEditR will foster further discoveries in this rapidly expanding field.

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

“…Such OT editing could have potentially deleterious impacts on cellular function in a therapeutic setting. To investigate OT RNA editing in multiplex edited T cells, we delivered coBE4 mRNA with and without our three optimal sgRNAs and measured RNA editing at 12, 24, and 48 h post electroporation using the program MultiEditR (https:// moriaritylab.shinyapps.io/multieditr/) 34 . We used a rational approach that accounts for transcripts with OT RNA editing observed in the original reports 32 , as well as the expression of these transcripts in activated T cells (DICE, dice-database.org) in order to select five candidates with a high probability of RNA editing ( Supplementary Fig.…”

“…PCR products were purified using the MinElute 96 UF PCR Purification Kit (Qiagen, Hilden, Germany) and sent for Sanger sequencing (Eurofins, Luxembourg). Percent editing was estimated using the program Multi-EditR (https://moriaritylab.shinyapps.io/multieditr/) 34 .…”

Programmable C‐to‐U RNA editing using the human APOBEC 3A deaminase

“…GFP signal offers a convenient and fast albeit indirect and preliminary readout of editing. In contrast, sequencing of the reporter transcript enables direct and reliable quantifications of editing, with Sanger sequencing producing results generally consistent with that obtained using next‐generation sequencing (NGS; Sharma et al , 2016, 2017), especially when the chromatograms are analyzed with the EditR method (Kluesner et al , 2019).…”

“…For Sanger sequencing, the cells were lysed and the mRNA in the lysate reverse transcribed as described (Joung et al, 2017). The amplicons were then sequenced, and editing efficiencies determined using EditR, an "accurate, fast, and low-cost method for the identification and quantification of base editing from fluorescent Sanger sequencing data" (Kluesner et al, 2018(Kluesner et al, , 2019. For NGS, the cDNA was subjected to two rounds of PCR to add Illumina adaptors and sample barcodes using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs).…”

Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

“…Peak heights can be measured automatically using a peak-calling program such as BioEdit or Chromas [ 36 ]. An automated tool is currently under development, based on an easy validation method for detecting and quantifying RNA editing from Sanger sequencing ( Table 1 , [ 32 ]). However, users are required to manually trim the 5′ and 3′ ends of the trace file to reduce noisy sequencing.…”