Cytosine base editing systems with minimized off-target effect and molecular size

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Cytosine base editing allows precise point mutations without DNA double-strand breaks, crucial for gene therapy.
• Existing base editing systems require improvements in reducing off-target mutations and enhancing delivery efficiency.

Purpose of the Study:

• To engineer the Target-AID cytosine base editing system for minimized off-target effects and reduced molecular size.
• To develop a base editing system suitable for Adeno-Associated Virus (AAV) vector delivery.

Main Methods:

• Structure-based rational engineering of the deaminase component (PmCDA1) of Target-AID.
• Truncation of the DNA-binding domain and introduction of mutations to restore enzyme function, creating tCDA1EQ.
• Fusion and integration of tCDA1EQ with Cas9 (AID-2S and AID-3S architectures) and SaCas9 for base editing in human cells.

Main Results:

• Engineered tCDA1EQ demonstrated minimized RNA-mediated editing and both gRNA-dependent and independent DNA off-targets.
• Fusion with the smaller SaCas9 resulted in a cytosine base editing system fitting within the AAV vector size constraints.
• The modified system (AID-2S and AID-3S) showed enhanced specificity and reduced collateral editing.

Conclusions:

• Rational engineering of Target-AID significantly reduces off-target activity and molecular size.
• The developed base editing system is compatible with SaCas9 and AAV delivery, paving the way for improved in vivo gene editing therapies.
• This work provides a more precise and deliverable tool for therapeutic applications requiring targeted DNA modification.