TadA reprogramming to generate potent miniature base editors with high precision

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Miniature CRISPR-Cas12f systems offer potential for gene editing but their base editor derivatives (miniCBEs and miniABEs) require further investigation.
• Functional miniature cytosine base editors (miniCBEs) have not been established, limiting their application.

Purpose of the Study:

• To develop and characterize novel Cas12f-derived miniature base editors with enhanced editing activity and broader targeting capabilities.
• To engineer highly precise miniCBEs with minimized off-target effects by improving deaminase components.

Main Methods:

• Generation of various Cas12f-derived miniCBEs and miniABEs.
• Engineering of TadA deaminase through mutagenesis screening for improved precision.
• Assessment of editing efficacy, targeting range, and off-target effects in vitro.
• In vivo delivery of miniCBEs and miniABEs via adeno-associated virus in the brain.

Main Results:

• Cas12f-derived miniCBEs and miniABEs demonstrated improved editing activities and expanded targeting scopes.
• Traditional cytidine deaminases in miniCBEs resulted in wide editing windows but significant off-target effects.
• Engineered TadA deaminase yielded potent miniCBEs with high precision and reduced off-target mutations.
• Newly designed miniCBEs and miniABEs successfully corrected pathogenic mutations in cell lines and induced genetic mutations in vivo.

Conclusions:

• This study presents novel strategies for developing cytosine base editors (CBEs) and expands the toolkit of miniature base editors.
• The developed miniCBEs and miniABEs offer promising therapeutic potential for clinical applications in gene therapy.