Selict-seq profiles genome-wide off-target effects in adenosine base editing

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Adenosine base editors (ABEs) are powerful tools for precise A·T to G·C conversions, crucial for treating genetic diseases.
• Unlike CRISPR-Cas9, ABEs avoid double-strand breaks, but their genome-wide off-target effects require thorough characterization.

Purpose of the Study:

• To develop and validate a biochemical method, Selict-seq, for comprehensively evaluating ABE genome-wide off-target editing.
• To elucidate the off-target editing landscape induced by ABEs, including previously unreported mutation sites.

Main Methods:

• Development of Selict-seq, a biochemical assay to capture deoxyinosine-containing single-stranded DNA.
• Identification of deoxyadenosine-to-deoxyinosine (dA-to-dI) mutation sites to map ABE off-target activities.
• Application of Selict-seq to analyze off-target edits for three single-guide RNAs, including the ABE8e(V106W) variant.

Main Results:

• Selict-seq successfully identified numerous unexpected off-target edits induced by ABEs, both within and outside protospacer regions.
• The ABE8e(V106W) editor demonstrated distinct off-target profiles, with high editing rates at novel sites like RNF2 and EMX1.
• Identified out-of-protospacer mutations, expanding the known off-target profile of ABEs.

Conclusions:

• Selict-seq provides an unbiased and widely applicable method for analyzing the ABE editome and evaluating genome editing tool specificity.
• This study significantly enhances the understanding of ABE off-target effects, crucial for their safe therapeutic application.
• The findings pave the way for improved specificity assessments of emerging deamination-based genome editing technologies.