RNA Editing pp 51-67 | Cite as

C-to-U RNA Editing: From Computational Detection to Experimental Validation

  • Taga Lerner
  • Mitchell Kluesner
  • Rafail Nikolaos Tasakis
  • Branden S. Moriarity
  • F. Nina Papavasiliou
  • Riccardo PecoriEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2181)


The AID/APOBEC family of enzymes are cytidine deaminases that act upon DNA and RNA. Among APOBECs, the best characterized family member to act on RNA is the enzyme APOBEC1. APOBEC1-mediated RNA editing plays a key role in lipid metabolism and in maintenance of brain homeostasis. Editing can be easily detected in RNA-seq data as a cytosine to thymine (C-to-T) change with regard to the reference. However, there are many other sources of base conversions relative to reference, such as PCR errors, SNPs, and even DNA editing by mutator APOBECs. Furthermore, APOBEC1 exhibits disparate activity in different cell types, with respect to which transcripts are edited and the level to which they are edited. When considering these potential sources of error and variability, an RNA-seq comparison between wild-type APOBEC1 sample and a matched control with an APOBEC1 knockout is a reliable method for the discrimination of true sites edited by APOBEC1. Here we present a detailed description of a method for studying APOBEC1 RNA editing, specifically in the murine macrophage cell line RAW 264.7. Our method covers the production of an APOBEC1 knockout cell line using the CRISPR/Cas9 system, through to experimental validation and quantification of editing sites (where we discuss a recently published algorithm (termed MultiEditR) which allows for the detection and quantification of RNA editing from Sanger sequencing). Importantly, this same protocol can be adapted to any RNA modification detectable by RNA-seq analysis for which the responsible protein is known.

Key words

C-to-U RNA editing AID/APOBECs APOBEC1 RNA editing detection Validation and quantification CRISPR/Cas9 MultiEditR Sanger sequencing 



手机体育投注平台We thank the Flow Cytometry unit of the Imaging and Cytometry Core Facility, German Cancer Research Center (DKFZ), for providing excellent sorting services. We also thank Derek Nedveck (University of Minnesota) for his help with questions surrounding R shiny. Finally we also thank Walker Lahr (University of Minnesota) for helpful conversations surrounding this topic.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Taga Lerner
    • 1
    • 2
  • Mitchell Kluesner
    • 3
    • 4
    • 5
  • Rafail Nikolaos Tasakis
    • 1
    • 2
  • Branden S. Moriarity
    • 3
    • 4
    • 5
  • F. Nina Papavasiliou
    • 1
  • Riccardo Pecori
    • 1
    Email author
  1. 1.Division of Immune Diversity, Program in Cancer ImmunologyGerman Cancer Research Center (DKFZ)HeidelbergGermany
  2. 2.Faculty of BiosciencesHeidelberg UniversityHeidelbergGermany
  3. 3.Department of PediatricsUniversity of MinnesotaMinneapolisUSA
  4. 4.Center for Genome Engineering, University of MinnesotaMinneapolisUSA
  5. 5.Masonic Cancer Center, University of MinnesotaMinneapolisUSA

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