Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• Current DNA base editors primarily target cytosine (C) and adenine (A) bases.
• Existing technologies lack efficient methods for guanine (G) or thymine (T) base editing.
• The development of novel base editing tools is crucial for expanding genome engineering capabilities.

Purpose of the Study:

• To develop a novel base editor capable of editing guanine (G) bases.
• To engineer a deaminase-free glycosylase-based editor for precise G base modifications.
• To demonstrate the efficacy and efficiency of the new guanine base editor (gGBE).

Main Methods:

• Fusion of Cas9 nickase with an engineered N-methylpurine DNA glycosylase (MPG).
• MPG mutagenesis and screening using an intron-split EGFP reporter system.
• Testing gGBE efficiency in cultured human cells and mouse embryos.

Main Results:

• Engineered MPG significantly increased G editing efficiency by over 1500-fold.
• gGBE achieved high base editing efficiency (up to 81.2%).
• High G-to-T or G-to-C (G-to-Y) conversion ratios (up to 0.95) were observed.

Conclusions:

• A novel deaminase-free glycosylase-based guanine base editor (gGBE) has been successfully developed.
• The gGBE system demonstrates a proof-of-concept for a new base editing approach targeting guanine.
• This advancement expands the toolkit for precise genome editing, particularly for guanine modifications.