One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.
Although it is known that blood vessels undergo remodeling in type 2 diabetes (T2D), the signaling pathways that underlie the structural and functional changes seen in diabetic arteries remain unclear. Our objective was to determine whether the remodeling in type 2 diabetic Goto-Kakizaki (GK) rats is evoked by elevated reactive oxygen species (ROS). Our results show that aortas from GK rats produced greater force (P < 0.05) in response to stimulation with KCl and U46619 than aortas from Wistar rats. Associated with these changes, aortic expression of contractile proteins (measured as an index of remodeling) and the microRNA (miR-145), which act to upregulate transcription of contractile protein genes, was twofold higher (P < 0.05) in GK than Wistar (age-matched control) rats, and there was a corresponding increase in ROS and decrease in nitric oxide signaling. Oral administration of the antioxidant Tempol (1 mmol/l) to Wistar and GK rats reduced (P < 0.05) myocardin and calponin expression. Tempol (1 mmol/l) decreased expression of miR-145 in Wistar and GK rat aorta. To elucidate the mechanism through which ROS increases miR-145, we measured their levels in freshly isolated aorta and cultured aortic smooth muscle cells incubated for 12 h in the presence of H2O2 (300 μmol/l). H2O2 increased expression of miR-145, and there were corresponding nuclear increases in myocardin, a miR-145 target protein. Intriguingly, H2O2-induced expression of miR-145 was decreased by U0126 (10 μmol/l), a MEK1/2 inhibitor, and myocardin was decreased by anti-miR-145 (50 nmol/l) and U0126 (10 μmol/l). Our novel findings demonstrate that ROS evokes vascular wall remodeling and dysfunction by enhancing expression of contractile proteins in T2D.
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