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A Nonsequencing Approach for the Rapid Detection of RNA Editing
Published on: April 21, 2022
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APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels.
Amanda E Rieffer1, Yanjun Chen2, Daniel J Salamango1
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota-Twin Cities, Minneapolis, Minnesota, USA; University of Texas Health San Antonio, San Antonio, Texas, USA.
The CRISPR Journal
|September 6, 2023
Summary
A new tool, the APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels (ARSENEL), enables real-time monitoring of base editor performance across all dinucleotide motifs. This system aids in developing more precise genome engineering technologies.
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.
