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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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Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors
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Off-target RNA editing hotspots caused by base editors.

Michelle Shmuel-Eidelman1, Roni Cohen-Fultheim1, Eli Eisenberg2

  • 1Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel; The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 5290002, Israel.

Molecular Therapy : the Journal of the American Society of Gene Therapy
|December 25, 2025
PubMed
Summary
This summary is machine-generated.

This study introduces a new method to detect unintended RNA edits from base editors, a gene-editing tool. It identifies specific genes and regions prone to these off-target edits, crucial for developing safer gene therapies.

Keywords:
RNA editing indexadenine base editorsbase editorscytosine base editorsgenome editingoff-target activity

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Base editors offer precise genome and transcriptome modification for therapeutic applications.
  • Clinical translation of base editing is limited by off-target, unintended edits.
  • Current methods primarily detect recurrent RNA deaminations, missing stochastic off-target events.

Purpose of the Study:

  • To develop and apply a novel methodology for quantifying the total burden of RNA alterations induced by base editors.
  • To identify specific genes and genomic regions susceptible to off-target base editing.
  • To enhance understanding of base editor specificity and guide the development of safer gene-editing tools.

Main Methods:

  • Application of the RNA editing index algorithm to quantify off-target RNA alterations.
  • Systematic analysis of off-target editing levels across individual genes and exon regions.
  • Identification of hotspot genes and localized regions prone to aberrant base editing.

Main Results:

  • Quantification of off-target RNA editing levels across genes using the RNA editing index.
  • Identification of 2,844 adenine base editor and 1,253 cytosine base editor hotspot genes.
  • Discovery of localized exon regions susceptible to off-target editing, including those introducing premature stop codons.

Conclusions:

  • The developed methodology effectively captures stochastic off-target edits missed by conventional detection.
  • Understanding off-target landscapes is critical for assessing therapeutic risks, such as premature stop codon introduction.
  • This study provides a framework for improving base editor precision and accelerating the development of safer gene-editing technologies.