A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA), efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.
High-throughput Sequencing data have been deposited in GEO under accession codes GSE146999. Source data files have been provided as excel files.
Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophyNCBI Gene Expression Omnibus, GSE146999.
- Balamurali K Ambati
- Balamurali K Ambati
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#15-11024 and #18-10016) of the University of Utah.
- Constance L Cepko, Harvard Medical School, United States
© 2021, Uehara et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Two epigenetic pathways of transcriptional repression, DNA methylation and Polycomb repressive complex 2 (PRC2) are known to regulate neuronal development and function. However, their respective contributions to brain maturation are unknown. We found that conditional loss of the de novo DNA methyltransferase Dnmt3a in mouse excitatory neurons altered expression of synapse-related genes, stunted synapse maturation, and impaired working memory and social interest. At the genomic level, loss of Dnmt3a abolished postnatal accumulation of CG and non-CG DNA methylation, leaving adult neurons with an unmethylated, fetal-like epigenomic pattern at ~222,000 genomic regions. The PRC2-associated histone modification, H3K27me3, increased at many of these sites. Our data support a dynamic interaction between two fundamental modes of epigenetic repression during postnatal maturation of excitatory neurons, which together confer robustness on neuronal regulation.
A single Dcp1-Dcp2 decapping enzyme targets diverse classes of yeast mRNAs for decapping-dependent 5' to 3' decay, but the molecular mechanisms controlling mRNA selectivity by the enzyme remain elusive. Through extensive genetic analyses we reveal that Dcp2 C-terminal domain cis-regulatory elements control decapping enzyme target specificity by orchestrating formation of distinct decapping complexes. Two Upf1-binding motifs direct the decapping enzyme to NMD substrates, a single Edc3-binding motif targets both Edc3 and Dhh1 substrates, and Pat1-binding leucine-rich motifs target Edc3 and Dhh1 substrates under selective conditions. Although it functions as a unique targeting component of specific complexes, Edc3 is a common component of multiple complexes. Scd6 and Xrn1 also have specific binding sites on Dcp2, allowing them to be directly recruited to decapping complexes. Collectively, our results demonstrate that Upf1, Edc3, Scd6, and Pat1 function as regulatory subunits of the holo-decapping enzyme, controlling both its substrate specificity and enzymatic activation.