How DNA Repair Proteins Influence Base Editing Efficiency

A recent study led by Alexis Komor from the University of California San Diego, and published in Nature Communications, investigates how DNA repair proteins affect the outcomes of base editing—a gene-editing technique developed by Komor. Base editing enables precise chemical changes to DNA bases, offering an alternative to traditional CRISPR-Cas9 editing, which requires double-stranded DNA breaks and can result in unwanted insertions or deletions.

Komor’s team focused on cytosine base editors (CBEs), which convert cytosine (C) to thymine (T) without cutting both DNA strands. While CBEs are efficient, the group sought to understand the cellular processes that influence editing outcomes. “We know the base editor works, but how?” That’s the main question Komor’s group wanted to answer in this study.

To explore this, the researchers used gene knockdown techniques to reduce the expression of each of the 2,015 DNA repair or processing proteins in the human genome. They then used green fluorescent protein markers to identify cells with successful C to T edits and analyzed which proteins’ absence improved or hindered editing.

The study found that the presence of uracil N-glycosylase (UNG) increased unwanted editing outcomes, while inhibiting UNG boosted CBE efficiency. Another key finding was that the ligase Lig3 reduced CBE editing by sealing DNA nicks before the desired conversion could occur. “We think that the Lig3 can sneak in and seal that nick back together. Then we don't have a chance for that uracil to be turned into a thymine anymore,” Komor explained. Conversely, the mismatch repair pathway, particularly the MutS-alpha complex, aided the conversion process.

Komor emphasizes that understanding these mechanisms is crucial for improving base editor efficiency and safety. “These base editing tools are good, but they’re not perfect,” she stated.