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Toward optimizing diversifying base editors for high-throughput mutational scanning studies
Carley I Schwartz1,2, Nathan S Abell3, Amy Li3
1Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, United States.
Nucleic Acids Research
|July 4, 2025
Summary
Diversifying base editors (DivA-BE) efficiently install point mutations in mammalian genomes. Direct fusion of deaminase to dCas9 N-terminus maximizes editing efficiency for functional variant discovery.
Area of Science:
- Genetics
- Molecular Biology
- Bioengineering
Background:
- Base editors are powerful tools for studying gene function by introducing specific mutations.
- Limited understanding exists on how base editor design impacts editing outcomes.
Purpose of the Study:
- To investigate how deaminase fusion architecture and delivery methods affect base editing efficiency.
- To identify optimal base editor designs for systematic genomic mutation introduction.
Main Methods:
- Assessed editing efficiency of various base editor designs across ~200 synthetic target sites.
- Compared N-terminal vs. C-terminal deaminase fusion to dCas9 (DivA-BE).
- Evaluated delivery methods including electroporation and lentiviral transduction.
Main Results:
- Direct fusion of hyperactive deaminase to the N-terminus of dCas9 (DivA-BE) yielded the highest editing efficiency, approximately 4-fold greater than previous methods.
- DivA-BE preferentially mutagenized the target strand, creating C>N mutations.
- Increased indel frequencies were observed with DivA-BE compared to other designs.
Conclusions:
- DivA-BE editors demonstrate superior efficiency for diversifying target sites in mammalian genomes.
- The N-terminal fusion architecture is optimal for maximizing base editing efficiency and strand-specific mutagenesis.
- DivA-BE editors are well-suited for mutational scanning assays to discover functional genetic variants.
