Suppressing gain-of-function proteins via CRISPR/Cas9 system in SCA1 cells.

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.

Molecular characterization of toxigenic Clostridium difficile in a northern Italian hospital.

Clostridium difficile is responsible for more than 90 % of cases of antibiotic-associated diarrhea and pseudomembranous colitis. The most important virulence factors are two toxins called enterotoxin A and cytotoxin B; some C. difficile strains contain the C. difficile binary toxin (CDT). The aim of our study was to prospectively analyze C. difficile clinical isolates in a single center to determine the molecular features of collected strains. Among the 252 isolates, 217 were A + B + (86.1 %), 33 were A + B + cdt + (13.1 %) and 2 were A - B + (0.8 %). There were 15 different ribotypes with a predominance of 018.