Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas base editing enables targeted DNA changes for research and therapy.
• Cytosine base editors (CBEs) utilize APOBEC1 for C-to-T conversions.
• Previous research noted off-target DNA edits by CBEs.

Purpose of the Study:

• To investigate potential off-target RNA editing by CBEs in human cells.
• To engineer CBE variants with reduced RNA editing activity.
• To assess the impact of RNA edits on gene function and protein products.

Main Methods:

• Utilized a rat APOBEC1-based CBE system in human cells.
• Performed transcriptome-wide analysis to identify C-to-U edits.
• Engineered and tested mutated APOBEC1 variants for reduced RNA editing.
• Evaluated on-target DNA editing efficiency and specificity of engineered variants.

Main Results:

• CBEs induced extensive transcriptome-wide C-to-U edits in human cells, affecting a large percentage of genes.
• RNA edits occurred in coding and non-coding regions, leading to various mutations.
• Engineered CBE variants showed >390-fold and >3,800-fold reduction in RNA edits.
• Mutant CBEs exhibited improved on-target DNA editing precision without significant efficiency loss.
• Adenine base editors were also found to induce transcriptome-wide RNA edits.

Conclusions:

• CBEs pose a risk of unintended, widespread RNA modifications.
• Engineering efforts can yield base editors with enhanced specificity and reduced RNA off-target effects.
• Thorough characterization of RNA off-target effects is crucial for base editor applications in research and clinics.