APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Cytosine base editors (CBEs) are advancing precision genome engineering.
• Current CBEs are primarily optimized for TC dinucleotide targets, limiting broader application.
• Evaluating editing across all dinucleotide motifs is crucial for developing versatile base editors.

Purpose of the Study:

• To develop and implement a novel reporter system, ARSENEL, for real-time evaluation of base editing across all four cytosine dinucleotide motifs (AC, CC, GC, TC).
• To quantitatively assess the editing efficiency and specificity of various CBEs and identify potential inhibitors.

Main Methods:

• Development of the ARSENEL panel, comprising four constructs that report editing via eGFP fluorescence accumulation.
• Testing of twelve established and novel base editors using the ARSENEL system.
• Real-time assessment of natural and synthetic APOBEC inhibitors.

Main Results:

• ARSENEL successfully quantifies editing rates for AC, CC, GC, and TC motifs in living cells.
• Established CBEs showed expected biases (APOBEC3Bctd to TC, AIDΔC to GC).
• A full-length APOBEC3B CBE demonstrated high TC specificity, while APOBEC3A exhibited high editing efficiency. The Epstein-Barr virus ribonucleotide reductase large subunit was identified as a potent inhibitor.

Conclusions:

• ARSENEL provides a versatile platform for quantitative, real-time assessment of base editor performance and inhibitor screening.
• The system facilitates the development of highly specific and efficient genome editing tools.
• ARSENEL has significant potential for advancing research and development in precision genome engineering.