Engineered CBEs based on Macaca fascicularis A3A with improved properties for precise genome editing.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Cytidine deaminases, such as rat APOBEC1 (rA1), are key components of cytosine base editors (CBEs) for C-to-T conversion.
• Human APOBEC3A (hA3A) has been shown to improve CBE properties compared to rA1.
• The utility of macaque APOBEC3A (mA3A) orthologs in CBEs remains unexplored.

Purpose of the Study:

• To develop and evaluate engineered CBEs utilizing macaque APOBEC3A (mA3A).
• To compare the performance of mA3A-based CBEs against existing rA1- and hA3A-based systems.
• To investigate the impact of specific protein motifs, like Ser-Val-Arg (SVR), on CBE activity and specificity.

Main Methods:

• Construction and testing of novel CBE variants incorporating mA3A.
• Comparative analysis of on-target efficiency and off-target DNA/RNA activity.
• Site-directed mutagenesis to assess the role of the SVR motif in BE4-mA3A and BE4-hA3A.

Main Results:

• BE4-mA3A and its variants demonstrated enhanced CBE properties compared to BE3-rA1 and BE4-rA1, with the exception of DNA off-target activity.
• Deletion of the SVR motif in BE4-mA3A significantly reduced both DNA and RNA off-target activities while improving editing accuracy, without affecting on-target efficiency.
• A chimeric BE4-hA3A with the SVR motif showed a ~50% increase in editing efficiency, with other properties remaining unchanged.

Conclusions:

• mA3A-based CBEs represent promising candidates for further optimization, offering potential advantages over rA1- and hA3A-based systems.
• The SVR motif plays a critical role in determining the intrinsic properties, particularly specificity and accuracy, of CBEs.
• Engineered CBEs utilizing mA3A and strategic motif modification offer novel tools for precise gene editing applications.