Silencing ATXN2 through CRISPR-mediated insertion of a premature STOP codon

• Autor(a) principal: Reis, Ricardo António Afonso
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.1/15533
• topic: Ataxia espinocerebelosa tipo 2; Neurodegeneração; Edição génica; Sistema crispr/cas9; Silenciamento génico

Spinocerebellar ataxia type 2 (SCA2) is a hereditary neurodegenerative disorder caused by expansion of CAG trinucleotide repeats present in the codifying region of the ATXN2 gene. The mutant ATXN2 product - a ataxin-2 protein with an expanded polyglutamine tract - displays a cytotoxic gain-of-function, leading to progressive neurodegeneration. No definitive therapy for this disease has yet been developed, but ATXN2 knock-down represents a promising approach. The versatility of the CRISPR-Cas system enables gene silencing at a genomic level, through an array of unprecedently diverse mechanisms. Cas nucleases guided by a single guide RNA (sgRNA) molecule produce modifications in precise regions of the DNA, and the system can be modified to direct transcriptional inhibitors such as the Krüppel-associated box domain (KRAB) to particular genetic loci. The aim of this study was to develop two ATXN2 silencing strategies, utilizing catalytically active Cas9 to knock-down the ATXN2 gene or, alternatively, a dCas9-KRAB fusion protein to pre-transcriptionally repress ATXN2 expression. One sgRNA targeting the codifying region of ATXN2 was cloned into a plasmid encoding Cas9, and another sgRNA targeting the ATXN2 promoter was cloned into a plasmid encoding dCas9-KRAB. Plasmids were transfected into human embryonic kidney (HEK) 293T cells and endogenous ATXN2 mRNA and protein levels were analyzed by qPCR and Western blot. Cas9/sgRNA did not alter ATXN2 mRNA levels in comparison to controls, contrasting with ataxin-2 protein levels that revealed a decrease tendency. dCas9-KRAB/sgRNA did not alter ATXN2 mRNA levels, but displayed a tendency for decreasing ataxin-2 protein levels, relative to non-transfected cells. Our study suggests that the CRISPR-Cas9 system may be adapted for ATXN2 gene silencing applications, but further improvements to our strategies will be necessary to produce significant alterations.