Cas12a Base Editors Induce Efficient and Specific Editing with Low DNA Damage Response

Area of Science:

• Molecular Biology
• Gene Editing
• Biotechnology

Background:

• Base editors (BEs) offer potential for correcting disease-causing point mutations.
• Cas9 nickase (nCas9)-derived BEs can cause unwanted DNA double-strand breaks and DNA damage response (DDR).

Purpose of the Study:

• To develop a novel base editor system that minimizes DNA damage and DDR.
• To enhance deamination efficiency and editing specificity compared to existing systems.

Main Methods:

• Conjugating catalytically dead Cas12a (dCas12a) with engineered human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) to create the BEACON system.
• Evaluating BEACON's DNA break induction, DDR activation (H2AX, ATM, ATR, p53), C-to-T editing efficiency, and RNA off-target mutations in mammalian cells.
• Assessing in vivo base editing in mouse embryos and F0 mice.

Main Results:

• dCas12a-conjugated BEs induced minimal DNA breaks and DDR.
• The BEACON system achieved efficient C-to-T editing comparable to AncBE4max with low DDR and minimal RNA off-target mutations.
• BEACON successfully induced in vivo base editing in mouse embryos, with targeted C-to-T conversions observed in F0 mice.

Conclusions:

• The BEACON system represents a promising advancement in base editing technology, offering enhanced efficiency and specificity while minimizing unwanted DNA damage and DDR.
• BEACON's ability to perform in vivo base editing in mammalian embryos opens new avenues for therapeutic applications in genetic diseases.