Harnessing A3G for efficient and selective C-to-T conversion at C-rich sequences.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Site-specific DNA base editing utilizes engineered Cas proteins and cytidine deaminases to modify target cytosine (C) bases within a guide RNA-defined window.
• The initial base editor, BE3, uses rat APOBEC1 (rA1) but exhibits non-specific deamination and off-transcriptome edits.
• While motif-selective editors like eA3A-BE3 (targeting TC) exist, editors for other motifs, especially in C-rich sequences, and those mitigating RNA off-target effects were needed.

Purpose of the Study:

• To develop novel base editors capable of targeting specific DNA sequence motifs beyond TC.
• To address and overcome the off-target RNA editing issues associated with the original BE3 system.
• To enhance the precision and efficiency of DNA base editing, particularly in challenging C-rich genomic regions.

Main Methods:

• Engineered a new base editor by replacing the rA1 deaminase in BE3 with an optimized human APOBEC3G (oA3G).
• Developed oA3G-BE3 and an enhanced variant, oA3G-BE4max, to improve on-target editing efficiency and selectivity.
• Evaluated the on-target and off-target editing profiles of the new editors at both the DNA and transcriptome levels.

Main Results:

• The developed oA3G-BE3 selectively targets DNA sequences containing CC and CCC motifs.
• oA3G-BE4max demonstrated robust on-target editing efficiency and was free from global off-target effects on the transcriptome.
• Negligible Cas9-independent off-target edits were observed genome-wide for oA3G-BE4max.

Conclusions:

• oA3G-BE4max effectively edits C(C)C motifs with high precision and selectivity.
• This new editor complements existing eA3A-editors, broadening the scope of motif-selective base editing.
• The developed editors fill a critical gap in the base editing toolbox, enabling targeted modifications in previously challenging sequences.