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In vitro Mutagenesis

In vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

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Updated: May 28, 2026

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

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Toward optimizing diversifying base editors for high-throughput mutational scanning studies.

Carley I Schwartz^1,2, 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

This summary is machine-generated.

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.

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Last Updated: May 28, 2026

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Published on: February 17, 2023

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.