Background

Since the advent of CRISPR-Cas9, base editing technology, which modifies single bases without cleaving double-stranded DNA, has emerged as a key tool for treating genetic diseases. In particular, adenine base editors (ABEs), which substitute adenine with guanine, have garnered attention for their potential to correct mutations that cause rare genetic diseases.

However, existing technologies frequently suffer from the side effect of 'bystander editing,' where adenine in the vicinity of the target is also substituted. This is because the adenine deaminase (TadA) used in existing tools indiscriminately acts on all adenine within the target region. This can cause unwanted mutations and cellular toxicity, so there has been a continuous demand for technology that selectively corrects only single bases.

Key Discovery

The research team led by Professor Bae Sang-soo of the Department of Biochemistry at Seoul National University College of Medicine, in collaboration with Professor Kim Yo-han's team at Sungkyunkwan University, devised a new approach that applies RNA editing enzymes to DNA correction. The researchers focused on adenosine deaminase acting on RNA (ADAR), which converts RNA adenine to inosine in cells. ADAR exhibits the characteristic of selectively editing regions where base mismatches occur in double-stranded RNA. The researchers constructed a gene editing platform by combining nCas9-H840A with the active region of ADAR.

The platform, named snuABE, uses DNA:RNA hybrid double strands, formed by the binding of DNA and RNA, as the substrate for ADAR. To selectively edit only the target adenine, the researchers designed a unique target-adenine guide RNA (tagRNA). The tagRNA has a structure that artificially induces a base mismatch (mainly adenine-cytosine mismatch) at the adenine site to be corrected.

Thanks to this mismatch structure, the ADAR enzyme can recognize and correct only a single target adenine to guanine. Adenines in the surrounding area form normal complementary bonds and are excluded from enzyme activity. As a result, precise editing was achieved with almost no bystander sequence substitution errors.

The researchers found that among the ADARs of various organisms, the enzyme derived from human head lice (Pediculus humanus) was the most active. To improve editing efficiency, the enzyme was further modified using EvolvePro, an AI-based protein evolution algorithm. In addition, the 3'-terminal structure of tagRNA was chemically protected to secure intracellular stability.

snuABE showed an average editing efficiency of 5.4% and a maximum of 50.0% in experiments using the human embryonic kidney 293T (HEK293T) cell line. In particular, it showed excellent precision with almost no bystander editing errors, unlike existing tools. This is because it accurately edited only the target base even in harsh conditions where adenine was densely packed around the target. As a result of analyzing the genome-wide off-target effects, mutations were below the detection limit.

Significance and Prospects

This research is evaluated as expanding the paradigm of adenine base editing. It successfully transplanted the natural ADAR enzyme into the DNA correction field, breaking away from the existing TadA enzyme-based platforms. It is cited as a representative case that shows how the fusion of unique ideas and protein engineering technology can create new therapeutic tools.

However, further improvements are required before snuABE can be introduced into actual clinical practice. The average editing efficiency is relatively lower than existing platforms, so it needs to be further improved to ensure its practicality as a therapeutic agent. The possibility of inducing unintended RNA editing (RNA off-target) by targeting RNA in cells is also a challenge to be addressed. The researchers plan to overcome these limitations by conducting additional modifications.