C-to-U RNA editing: From computational detection to experimental validation

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.