Biology subjects

O'Hara, M.

Publications and source records attributed to O'Hara, M..

3 recordsLinked to original sources

bioRxiv2024

Low RT-based Genome Editing Fidelity in Mouse Hepatocytes: Challenges and Solutions

Abstract/SummaryIntegrase-mediated Programmable Genomic Integration (I-PGI) uses a Cas9 nickase (nCas9) with a reverse transcriptase (RT), to write a large serine integrase (LSI) target site (attB/P, here called "beacon") in a programmed location. Co-delivery of the LSI and a DNA template containing the cognate recognition site results in precise integration of the template in a specific genomic location. While we were able to achieve high-fidelity beacon placement in a range of primate cycling and non-dividing cells, when translating our technology into an in vivo rodent model (liver) we surprisingly observed very low beacon fidelity, with the vast majority of beacons being unsuitable for integration. This phenomenon was independent of mouse strain, but was specific to non-dividing cells, as a cycling mouse hepatocyte cell line (Hepa1-6) demonstrated very high levels of fidelity. To address this issue we utilized neonatal mice, which have a much higher proportion of proliferating hepatocytes than adult mice. This resulted in a significant increase in the placement of high-fidelity beacons, and achieved functional gene expression after I-PGI in a therapeutically relevant target site. In an alternate approach, we engineered transgenic mice with intact beacons placed in specific genomic locations, allowing us to optimize integrase and DNA template dosing and kinetics. In summary, we have identified a previously undescribed challenge when using RT-based editing to write long sequences (~40 bp) in non-dividing rodent hepatocytes. This phenomenon was specific to rodents and was not observed in primate dividing or non-dividing cells. This previously unidentified challenge using RTs will limit the use of I-PGI in mouse models, however here we describe two methods that address this issue.

bioengineering↗

bioRxiv2020

Transcriptome-Wide Off-Target Effects of Steric-Blocking Oligonucleotides

Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to mRNA and blocking access from cellular machinery such as splicing factors. SBOs have the potential to bind to near-complementary sites in the transcriptome, causing off-target effects. In this study, we used RNA-seq to evaluate the off-target differential splicing events of 81 SBOs and differential expression events of 46 SBOs. Our results suggest that differential splicing events are predominantly hybridization-driven, while differential expression events are more common and driven by other mechanisms. We further evaluated the performance of in silico screens for off-target events, and found an edit distance cutoff of three to result in a sensitivity of 14% and false discovery rate of 99%. A machine learning model incorporating splicing predictions substantially improved the ability to prioritize low edit distance hits, increasing sensitivity from 4% to 26% at a fixed FDR. Despite these large improvements in performance, the approach does not detect the majority of events at a false discovery rate below 99%. Our results suggest that in silico methods are currently of limited use for predicting the off-target effects of SBOs.

molecular biology↗

bioRxiv2019

ATP7B Variant c.1934T>G p.Met645Arg Causes Wilson Disease by Promoting Exon 6 Skipping

Wilson Disease is a recessive genetic disorder caused by pathogenic loss-of-function variants in the ATP7B gene. It is characterized by disrupted copper homeostasis resulting in liver disease and/or neurological abnormalities. The variant NM_000053.3:c.1934T>G (Met645Arg) has been reported as compound heterozygous and is highly prevalent among Wilson Disease patients of Spanish descent. Accordingly, it is classified as pathogenic by leading molecular diagnostic centers. However, functional studies suggest that the amino acid change does not alter protein function, leading one ClinVar submitter to question its pathogenicity. Here we used a minigene system and gene-edited HepG2 cells to demonstrate that c.1934T>G causes approximately 70% skipping of exon 6. Exon 6 skipping results in frameshift and stop gain, which is expected to cause loss of ATP7B function. The elucidation of the mechanistic effect for this variant resolves any doubt about its pathogenicity and enables the development of genetic medicines for restoring correct splicing.

genetics↗