Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Base editors are promising tools for correcting pathogenic mutations in research and therapeutics.
• Developing adenine transversion editors, particularly for A•T-to-C•G changes, has been a significant challenge in the field.

Purpose of the Study:

• To engineer novel base editors capable of efficient and precise adenine transversion, specifically A•T-to-C•G editing.
• To expand the capabilities of base editing technology for genetic correction and research applications.

Main Methods:

• Fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e.
• Laboratory evolution of mAAG to enhance efficiency and targeting scope.
• Engineering of adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant.

Main Results:

• The developed base editors catalyzed adenosine transversion in specific sequence contexts.
• Laboratory evolution increased A-to-C/T conversion efficiency up to 73% and broadened targeting.
• ACBEs precisely installed A-to-C transversions with minimal off-targeting effects.
• ACBEs demonstrated high-efficiency correction of pathogenic mutations in preclinical models (mouse embryos, human cell lines) and founder mice.

Conclusions:

• Adenine transversion editors, including ACBEs, significantly expand the capabilities of base editing.
• These editors offer precise A•T-to-C•G editing, holding promise for basic research and therapeutic development.