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A/C Simultaneous Conversion Using the Dual Base Editor in Human Cells.

Xiaohui Zhang^1,2,3, Yuting Guan^2,3, Dali Li^4,5

¹Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medial College, Beijing, China.

Methods in Molecular Biology (Clifton, N.J.)

|January 2, 2023

Summary

This summary is machine-generated.

Scientists developed a dual base editor (A&C-BEmax) that can simultaneously convert both adenine and cytosine bases in human cells. This new tool expands precise base editing capabilities for genetic research.

Keywords:

Adenine deaminase Base editing Cas9 Cytosine

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Area of Science:

Molecular Biology
Gene Editing Technologies
Biotechnology

Background:

Cytidine base editors (CBEs) and adenine base editors (ABEs) enable precise DNA modifications.
Existing base editors are limited to editing either adenine or cytosine bases exclusively.
The need for tools capable of simultaneous A-to-G and C-to-T conversions is critical in genetic research.

Purpose of the Study:

To develop and describe a protocol for a novel dual base editor, A&C-BEmax.
To demonstrate the simultaneous editing of both adenine (A) and cytosine (C) bases within the same DNA allele in human cells.
To provide a comprehensive workflow for utilizing A&C-BEmax and analyzing its outcomes.

Main Methods:

Fusion of cytidine and adenosine deaminases to a catalytically inactive Cas9 (Cas9n) to create the A&C-BEmax dual base editor.
Application of the A&C-BEmax system in HEK293T human cells for dual base editing experiments.
Utilization of BE-analyzer software for the quantitative analysis of dual base editing efficiency and outcomes.

Main Results:

The A&C-BEmax system successfully achieved simultaneous C•G to T•A and A•T to G•C base conversions in human cells.
Simultaneous A/C base conversion within the same allele was observed at frequencies up to 30%.
A complete experimental workflow, including data analysis, was established and can be completed within 2-3 weeks.

Conclusions:

A&C-BEmax represents a significant advancement in base editing technology, enabling dual adenine and cytosine base conversions.
The developed protocol provides a standardized method for employing A&C-BEmax in human cells, facilitating complex genetic modifications.
This dual base editing tool broadens the scope of precise genome engineering applications.