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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
Published on: November 3, 2020
Suppressing gain-of-function proteins via CRISPR/Cas9 system in SCA1 cells
Mariangela Pappadà1, Ottavia Bonuccelli1, Mattia Buratto1
1Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, via Fossato di Mortara 64/B, 44121, Ferrara, Italy.
Abstract:
SCAs are autosomal dominant neurodegenerative disorders caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. There are no treatments available to delay the onset, stop or slow down the progression of these pathologies. In this work we focus our attention on SCA1 which is one of the most common genotypes circulating in Italy. Here, we develop a CRISPR/Cas9-based approach to reduce both forms of the ATXN1 protein, normal and mutated with expanded polyQ. We started with the screening of 10 different sgRNAs able to target Exon 8 of the ATXN1 gene. The two most promising sgRNAs were validated in fibroblasts isolated from SCA1 patients, following the identification of the best transfection method for this type of cell. Our silencing approach significantly downregulated the expression of ataxin1, due to large deletions and the introduction of small changes in the ATXN1 gene, evidenced by NGS analysis, without major effects on cell viability. Furthermore, very few significant guide RNA-dependent off-target effects were observed. These preliminary results not only allowed us to identify the best transfection method for SCA1 fibroblasts, but strongly support CRISPR/Cas9 as a promising approach for the treatment of expanded polyQ diseases. Further investigations will be needed to verify the efficacy of our silencing system in SCA1 neurons and animal models.
Insights
Researchers developed a CRISPR/Cas9 gene-editing tool to reduce toxic ATXN1 protein levels in Spinocerebellar Ataxia type 1 (SCA1). This approach shows promise for treating polyQ diseases by targeting the mutated ATXN1 gene.
Area of Science:
- Genetics and Molecular Biology
- Neurodegenerative Diseases
- Gene Therapy
Background:
- Spinocerebellar Ataxias (SCAs) are inherited neurodegenerative disorders caused by toxic gain-of-function proteins with expanded polyglutamine (polyQ) tracts.
- Currently, no treatments exist to halt or slow the progression of SCAs.
- Spinocerebellar Ataxia type 1 (SCA1), a common SCA genotype, is caused by mutations in the ATXN1 gene.
Purpose of the Study:
- To develop and validate a CRISPR/Cas9-based gene-editing strategy to reduce both normal and expanded polyQ forms of the ATXN1 protein.
- To investigate the efficacy and safety of this approach in patient-derived cells for potential SCA1 treatment.
Main Methods:
- Screening of 10 single-guide RNAs (sgRNAs) targeting Exon 8 of the ATXN1 gene.
- Validation of the most effective sgRNAs in fibroblasts from SCA1 patients.
- Next-Generation Sequencing (NGS) analysis to confirm gene editing and assess off-target effects.
Main Results:
- The CRISPR/Cas9 approach significantly downregulated ataxin1 expression through large deletions and small insertions in the ATXN1 gene.
- NGS analysis confirmed successful gene editing with minimal off-target effects and no significant impact on cell viability.
- The study identified an optimal transfection method for SCA1 patient fibroblasts.
Conclusions:
- CRISPR/Cas9 gene editing is a promising therapeutic strategy for reducing toxic ATXN1 protein in SCA1.
- This approach holds potential for treating other expanded polyQ neurodegenerative diseases.
- Further research is warranted to evaluate the system's efficacy in SCA1 neurons and animal models.
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