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CRISPR/Cas9-based silencing of the ATXN1 gene in Spinocerebellar ataxia type 1 (SCA1) fibroblasts

Salvatori, F.; Pappadà, M.; Sicurella, M.; Buratto, M.; Simioni, V.; Tugnoli, V.; Marconi, P.

2020-07-04 neuroscience
10.1101/2020.07.04.187559 bioRxiv
Show abstract

Spinocerebellar Ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. Actually, there are no treatments available to delay the onset, stop or slow down the progression of this pathology. Many approaches developed over the years involve the use of siRNAs and antisense oligonucleotides (ASOs). Here we develop and validate a CRISPR/Cas9 therapeutic strategy in fibroblasts isolated from SCA1 patients. We started from the screening of 10 different sgRNAs able to recognize regions upstream and downstream the CAG repeats, in exon 8 of ATXN1 gene. The two most promising sgRNAs, G3 and G8, whose efficiency was evaluated with an in vitro system, significantly downregulated the ATXN 1 protein expression. This downregulation was due to the introduction of indels mutations into the ATXN1 gene. Notably, with an RNA-seq analysis, we demonstrated minimal off-target effects of our sgRNAs. These preliminary results support CRISPR/Cas9 as a promising approach for treated polyQ-expanded diseases.

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