Off-target RNA editing hotspots caused by base editors.

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.