Riassunto analitico
Genome editing is a revolutionary technology enabling precise changes in the genome. This technology exploits the formation of DNA double strand breaks (DSB) and the consequent DNA repair through homologous recombination (HR) or non-homologous end joining (NHEJ). As a consequence, gene editing can lead to gene knock-out (KO), gene addition or correction. The programmed endonucleases used in this technology are able to induce a site-specific DSB. Among the recently developed genome editing technologies, Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas)-system is particularly promising. The CRISPR/Cas9 system was adapted from Streptococcus pyogenes immune system. The engineered CRISPR/Cas9 system requires two components: the endonuclease Cas9 and the guide RNA (gRNA) that tethers the Cas9 to a specific sequence. Thanks to its feasibility and easy-to-use, CRISPR/Cas9 system has provided a new popular tool for accurate manipulation of genomic sequence. This thesis describes a CRISPR/Cas9-specific targeting of a heterozygous de novo mutation in COL6A1 gene, exerting a dominant negative effect on collagen VI assembly and secretion, that leads to Ullrich congenital myopathy disorder (UCMD), the most severe form of collagen VI-related myopathies. To date there is no curative therapy for this disease, despite few pharmacological therapeutic attempts currently underway or upcoming. A permanent down-regulation of the mutated COL6A allele represents a possible therapeutic intervention. Consequently, the aim of the thesis is to demonstrate that is possible to achieve a specific CRISPR/Cas9-mediated KO of the mutated allele in patient fibroblasts, resulting in a reduction of its expression. To specifically target the mutation identified in a UCMD patient (#2), two gRNAs were designed. In particular, gRNA2 is tailored to the mutated region of COL6A1 gene, localized in the junction between exon 8 and intron 8. On the contrary, gRNAint3 recognized a sequence in the intron 3 of COL6A1gene. Combining the activity of two gRNAs, two DSBs on the COL6A1 gene will be introduced in order to get the deletion of the sequence encompassing the mutation. The triggered NHEJ mechanism should result in the junction between exons 3 and 9, altering the open reading frame of the sequence and leading to the generation of a new stop codon in the exon 10 of COL6A1 mutated gene. Instead, occurring of a single DSB in the COL6A1 gene should not alter the frame of the mutated mature transcript as well as the wt one. The designed gRNAs were both cloned in plasmid expressing the humanized S. pyogenes Cas9 (hSpCas9) to obtain the effector plasmid. To assess the specificity of the target template plasmids bearing a wt or mutated fragment of the affected gene were generated. The effector and template plasmids were properly co-transfected in HEK293T cells. The cleavage efficiency on wt and mutated templates was verified by gel electrophoresis of COL6A1 templates amplified post editing. The double editing occurring on mutated template should generate a shorter fragment that could quantified by densitometric analysis. The cleavage occurring on genome-wide off-targets predicted by bioinformatic tools were assessed by Sanger sequencing and Tracking of Indels by Decomposition (TIDE) analysis. The CRISPR/Cas9 system was finally tested in fibroblasts derived from patient #2 and healthy donor. To this aim, a ribonucleoprotein (RNP) complex combining two gRNAs and the Cas9 protein was set up. The complex was then transfected into fibroblasts and the effect of double editing was verified at transcriptional level. A semiquantitative RT-PCR on mutated and control cells showed the presence of a shorter transcript corresponding to the elimination of the mutation causing UCMD. In conclusion, this thesis represents a proof-of- principle application of CRISPR/Cas9 system to selectively disrupt COL6A1 dominant mutations leading to UCMD.
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