Sensitive and unbiased genome-wide profiling of base-editor-induced off-target activity using CHANGE-seq-BE

Area of Science:

• Biotechnology
• Genomics
• Molecular Biology

Background:

• Assessing base editor safety is crucial for gene editing applications.
• Current methods for detecting off-target effects are limited by sensitivity and computational bias.

Purpose of the Study:

• To introduce CHANGE-seq-BE, a novel method for sensitive and unbiased detection of base editor off-target profiles.
• To evaluate the guide RNA-dependent off-target activity of adenine and cytosine base editors genome-wide.

Main Methods:

• CHANGE-seq-BE utilizes selective sequencing of in vitro base-editor-modified genomic DNA.
• The method comprehensively identifies genome-wide off-target mutations.
• Applied to genotoxicity studies for a CD40L-deficient patient receiving adenine base editor treatment.

Main Results:

• CHANGE-seq-BE provides a sensitive and unbiased assessment of off-target mutations.
• ABE8e adenine base editors showed substantially higher off-target activity (98.8% unique sites) compared to Cas9 nuclease.
• The method supported genotoxicity assessments for a novel gene therapy application.

Conclusions:

• CHANGE-seq-BE is a critical tool for characterizing base editor safety profiles.
• Base editor-specific methods are essential for accurate off-target activity identification.
• This technology advances the safety evaluation of gene editing tools for therapeutic use.