Biology subjects

Oh, Y. E.

Publications and source records attributed to Oh, Y. E..

4 recordsLinked to original sources

bioRxiv2025

Site-specific replacement of large-scale DNA fragments in human cells

Despite advances in genome editing1-4, precisely replacing large-scale fragments in human cells remains a significant challenge. Here, we present a site-specific gene replacement tool, named Prime Assembly (PA), which adapts prime editors to produce one or two pairs of 3-flaps on both the genome and donor plasmids. These 3-flaps anneal to each other precisely, similar to "Gibson Assembly" in DNA oligonucleotides5, allowing megabase-scale genomic excision and/or kilobase-scale donor insertion at the gene of interest. The PA system achieves an efficiency of up to 57.8% in replacing endogenous sequences with a 2.9 kbp donor DNA fragment in HEK293T cells, and presents an accuracy of more than 99% for integrated PA fragments. Ultimately, the site-specific replacement of large-scale coding sequences (CDSs) in disease-related genes can restore gene function across numerous patients with different mutations, providing a gene implantation technique for genome editing and a universal therapeutic approach.

molecular biology↗

bioRxiv2025

Engineered ADARs enable single-nucleotide resolution DNA A-to-G editing without bystander effects

The adenine base editor (ABE), which enables A*T-to-G*C base conversion, has emerged as a powerful tool for therapeutic applications. However, conventional ABEs suffer from bystander nucleotide conversions, limiting their utility for precise editing. Here, we present a single-nucleotide resolution ABE (snuABE) created by fusing a nickase Cas9, nCas9(H840A), with the deaminase domain of ADAR, which acts on DNA:RNA hybrids, instead of TadA, which acts on single-stranded DNA in conventional ABEs. snuABE requires a specially designed target-adenine guide RNA (tagRNA) that introduces a mismatch at the target adenine, enabling highly specific A-to-G editing by ADAR. Engineering ADAR from Pediculus humanus using the in silico protein evolution algorithm EvolvePro, along with 3-end protection of the tagRNA, further enhances the editing activity of snuABE in human cells. Moreover, snuABE exhibits significantly reduced DNA off-target activity, highlighting its potential as a safer and more precise base editing technology for therapeutic applications.

bioengineering↗

bioRxiv2024

AI-generated small binder improves prime editing

The prime editing 2 (PE2) system comprises a nickase Cas9 fused to a reverse transcriptase utilizing a prime editing guide RNA (pegRNA) to introduce desired mutations at target genomic sites. However, the PE efficiency is limited by mismatch repair (MMR) that excises the DNA strand containing desired edits. Thus, inhibiting key components of MMR complex through transient expression of a dominant negative MLH1 (MLH1dn) exhibited approximately 7.7-fold increase in PE efficiency over PE2, generating PE4. Herein, by utilizing a generative artificial intelligence (AI) technologies, RFdiffusion and AlphaFold 3, we ultimately generated a de novo MLH1 small binder (named MLH1-SB), which bind to the dimeric interface of MLH1 and PMS2 to disrupt the formation of key MMR components. MLH1-SBs small size (82 amino acids) allowed it to be integrated into pre-existing PE architectures via the 2A system, creating a novel PE-SB platform. Resultantly, by incorporating MLH1-SB into PE7, we have developed an improved PE architecture called PE7-SB, which demonstrates the highest PE efficiency to date (29.4-fold over PE2 and 2.4-fold over PE7 in HeLa cells), providing an insight that generative AI technologies will boost up the improvement of genome editing tools.

bioengineering↗

bioRxiv2024

Root traits and tree functional groups determine variability in root exudation and N uptake rates in mature temperate trees

While root exudation has the potential to affect soil biogeochemistry profoundly, the process is rarely quantified in mature, field-grown trees. We measured rates of carbon (C) exudation in 11 trees species that exhibit divergent root traits, including gymnosperms and angiosperms that associate with either arbuscular mycorrhizal (AM) or ectomycorrhizal (EcM) fungi. Our goal was to explore how tree species, plant functional groups and root traits collectively influence exudation patterns. Intraspecific variation in exudation rates was larger than interspecific variation, and neither functional groups nor morphological traits alone could sufficiently explain variation in this flux. EcM-associated gymnosperms exuded 2.4 times more C than EcM angiosperms and 1.5 times more than AM gymnosperms. Exudation rates correlated positively with specific root length (SRL) and specific root area (SRA), and were correlated with root tissue density and root diameter in EcM-associated species. Mixed-effect models revealed that exudation rates were best determined by a combination of phylogenetic group, tree-mycorrhizal type and SRA, though a large portion of unexplained variation suggests that contemporary environmental and local edaphic conditions are likely important. Collectively, our results reveal that exudation is a complex physiological process governed by multiple factors and cannot be fully explained by functional groups or root traits alone. Instead, a combined consideration of these factors and new experimental approaches may be needed before exudation patterns can be linked to plant trait frameworks and incorporated into large-scale models.

ecology↗