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CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice
Ralf S. Schmid, … , Camilo Breton, James M. Wilson
Ralf S. Schmid, … , Camilo Breton, James M. Wilson
Published January 7, 2021
Citation Information: J Clin Invest. 2021;131(5):e142574. https://doi.org/10.1172/JCI142574.
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Concise Communication Genetics Neuroscience Article has an altmetric score of 17

CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice

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Abstract

Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in CNS diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Because of genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in patients with AS that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain resulted in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrected the behavioral phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS because this may provide a lasting therapeutic effect for patients with AS.

Authors

Ralf S. Schmid, Xuefeng Deng, Priyalakshmi Panikker, Msema Msackyi, Camilo Breton, James M. Wilson

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Figure 1

In vivo gene editing of Ube3a-ATS causes indel formation and expression of Ube3a-YFP reporter.

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In vivo gene editing of Ube3a-ATS causes indel formation and expression ...
(A) Schematic mouse Ube3a genomic locus (adapted from ref. 11). The region targeted in this study by sgRNAs is indicated. IC, imprinting center; snoRNA, small-nucleolar RNA. (B) In vitro indel frequencies for screened sgRNAs. (C) Ube3am+/pYFP mice were injected with ATS-GE vector at indicated timepoints. After 3 weeks, amplicon sequencing with cortical samples revealed an average of 14.7% of cells with indels in neonatal injected pups; the indel frequency was less than 2.9% at all other time points. Nontargeting CRISRP/Cas9 or CRISPR/dCas9 resulted in indel formation in less than 0.2% (1-way ANOVA with Tukey’s pairwise comparison, n = 2–6 mice/group). (D) Indels persisted in Ube3am+/pYFP neonatal mice injected with ATS-GE vector (1-way ANOVA with Tukey’s pairwise comparison, n = 3–6 mice/group). (E and F) Representative Western blots for cortices from C demonstrate robust expression of paternal Ube3a-YFP when probed with YFP antibodies (E) or Ube3a antibodies (F). Relative quantifications normalized to actin are shown below each lane; green arrow in F demarcates the quantified Ube3a-YFP bands. NT, nontargeting. (G) Representative immunofluorescence staining for cortices from C shows Ube3a-YFP expression in neurons throughout the cortex (scale bar: 100 μm). (H) qPCR gene expression analysis with primers specific for the Ube3a-ATS transcript. We detected a significant 28% reduction in Ube3a-ATS expression for Ube3am+/pYFP gene-edited cortex samples (n = 3–15 mice/group, 1-way ANOVA with Tukey’s pairwise comparison). (I) qPCR gene expression analysis with primers specific for neighboring transcripts detected no differences in expression (n = 3–4 mice/group, 1-way ANOVA with Tukey’s pairwise comparison, P > 0.4). Means are shown with standard error; * P < 0.05, *** P < 0.001, **** P < 0.0001.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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