Programmable DNA pyrimidine base editing via engineered uracil-DNA glycosylase

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Current DNA base editing relies on engineered deaminases, which cannot directly modify thymine or guanine.
• Limitations exist in editing specific DNA bases, hindering therapeutic applications.

Purpose of the Study:

• To develop a novel DNA base editing technology capable of directly editing thymine.
• To engineer a uracil-DNA glycosylase (UNG) for efficient and specific thymine base editing.

Main Methods:

• Utilized translesion DNA synthesis pathway and engineered uracil-DNA glycosylase (UNG).
• Employed structure-based rational design, homologous protein exploration, and mutation screening to identify a Deinococcus radiodurans UNG mutant.
• Fused the engineered DrUNG protein with nickase Cas9 to create a thymine base editor (TBE).

Main Results:

• Achieved efficient thymine base editing at endogenous sites with up to 55% editing efficiency.
• Demonstrated minimal cellular toxicity and high editing specificity.
• Successfully restored IDUA enzyme activity in Hurler syndrome patient-derived cells.

Conclusions:

• Engineered UNG enables efficient and specific thymine base editing via TBEs.
• TBEs offer a low-toxicity approach for base editing with therapeutic potential for genetic diseases.
• This technology expands the scope of base editing beyond current deaminase-based methods.