NIB

Spinal Muscular Atrophy (SMA) is a progressive, fatal and hereditary muscle-nerve disease caused by the inadequate production of the SMN protein that enables motor neurons to function, as a result of mutations in the SMN1 and SMN2 genes. The most fatal and common type of SMA, which has many different types, is type1, which is formed as a result of mutations on the SMN1 gene. Compared to SMN2, it is known that SMN1 gene produces more healthy SMN protein. Among many different variants, the incidence and pathogenicity are high; The c.5C> G mutation in exon1 of the SMN1 gene located on chromosome 5 was corrected with CRISPR-Cas9 and primary editing technology. Electroporation method, which transfers into the cell by applying an electrical field, is used. The applied correction procedures were controlled by flow cytometry. For this purpose, pegRNA (primary regulatory guide RNA) was designed and combined with Cas9 enzyme and reverse transcriptase enzyme to form RNP (ribonucleoprotein). RNP was tested in HeLa and SKMC (Human skeletal muscle) cell lines with voltage and time variables to determine the most favorable conditions for the electroporation method. In addition, two different fluorescent proteins; Physical and chemical properties of the cells were determined by flow cytometry using GFP (green fluorescent protein) and mCherry (red fluorescent protein). It has been proven that the detected mutation was corrected and integrated into the cell by Western Blot Analysis technology. As a result of the applications, the point mutation in exon1 was corrected and the production of healthy SMN protein has increased.