CRISPR/Cas9 gene-editing in an in vitro model of a familial form of ALS

Abstract: Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the degeneration and death of both upper and lower motor neurons for which there is no cure currently available. The degeneration of motor neurons leads to the inability to carry out voluntary movement and death usually occurs 3-5 years after disease onset by respiratory failure. The most common cause of familial ALS is associated with mutations in the superoxide dismutase gene (SOD1). The SOD1 gene codes for an enzyme that is ubiquitously expressed and is involved in neutralizing reactive oxygen species. Mutations in the SOD1 gene lead to a toxic gain-of-function and astrocytes with mutated SOD1 has been shown to kill motor neurons by releasing toxic factors. In this project the gene-editing tool CRISPR/Cas9 was used to knock down SOD1 in forebrain neural progenitor cells (FB NPCs) using single guide RNAs (sgRNAs) to target the SOD1 gene specifically. The embryonic stem cell line RC17 and the induced pluripotent and patient-specific stem cell line SOD1L144P were differentiated to forebrain neural progenitor cells (FB NPCs) using a 16-day protocol. Lentivirus vectors were used to deliver the Cas9 protein, a sgRNA targeting either exon 2 (sgRNA:E2) or exon 3 (sgRNA:E3) and the green-fluorescent protein (GFP) as a reporter gene into the cells. The success of the differentiation was validated based on immunocytochemistry staining for the expression of FOXG1 and nestin, and the transduction on the presence of GFP. Staining for SOD1 was carried out to study the SOD1 knock-down on a protein level. The knock-down of SOD1 was also analysed based on mRNA expression using qPCR with a SOD1-specific primer. The results from the qPCR showed that the cells transduced with sgRNA:E2 and sgRNA:E3 had a reduced expression of SOD1 in both cell lines. In the case of sgRNA:E3 the reduction was more than four-fold in both cell lines.