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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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A Nonsequencing Approach for the Rapid Detection of RNA Editing
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C-to-U RNA Editing: From Computational Detection to Experimental Validation.

Taga Lerner1,2, Mitchell Kluesner3,4,5, Rafail Nikolaos Tasakis1,2

  • 1Division of Immune Diversity, Program in Cancer Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|July 31, 2020
PubMed
Summary
This summary is machine-generated.

APOBEC1 enzyme mediates RNA editing crucial for lipid metabolism and brain function. A new method using CRISPR/Cas9 and RNA-seq reliably identifies APOBEC1 editing sites in murine macrophages, distinguishing true edits from errors.

Keywords:
AID/APOBECsAPOBEC1C-to-U RNA editingCRISPR/Cas9MultiEditRRNA editing detectionSanger sequencingValidation and quantification

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The AID/APOBEC enzyme family comprises cytidine deaminases acting on DNA and RNA.
  • APOBEC1 is the best-characterized member acting on RNA, influencing lipid metabolism and brain homeostasis.
  • RNA editing by APOBEC1 appears as C-to-T changes in RNA-seq data, but distinguishing true edits from errors (PCR, SNPs, DNA editing) is challenging.

Purpose of the Study:

  • To present a reliable method for studying APOBEC1-mediated RNA editing.
  • To detail a protocol for generating an APOBEC1 knockout cell line using CRISPR/Cas9.
  • To enable accurate detection and quantification of APOBEC1 editing sites in murine macrophages.

Main Methods:

  • Generation of an APOBEC1 knockout murine macrophage cell line (RAW 264.7) via CRISPR/Cas9.
  • RNA sequencing (RNA-seq) comparison between wild-type and knockout samples.
  • Experimental validation and quantification of editing sites using the MultiEditR algorithm for Sanger sequencing data.

Main Results:

  • A detailed protocol for studying APOBEC1 RNA editing in RAW 264.7 cells is provided.
  • The method allows for reliable discrimination of true APOBEC1 editing sites from other base conversion sources.
  • The protocol is adaptable for studying other RNA modifications detectable by RNA-seq.

Conclusions:

  • A robust method combining CRISPR/Cas9 knockout and RNA-seq analysis enables precise APOBEC1 RNA editing site identification.
  • This approach overcomes challenges posed by technical variability and other biological sources of base conversions.
  • The described protocol offers a versatile framework for investigating various RNA modification events mediated by specific proteins.