CRISPR/Cas9-Mediated Correction of the SLC25A38 Mutation in a Patient With Sideroblastic Anemia.

backgroundThe SLC25A38 is one of the genes of the heme biosynthesis pathway and disruptive alterations therein cause autosomal recessive congenital sideroblastic anemia (CSA). Primary patient-derived fibroblasts can be used as a valuable source for disease modeling, cell therapy studies, and the generation of induced pluripotent stem cells. In addition, they are easy to isolate and grow rapidly in vitro. Therefore, effective gene correction in these cells could be an important step towards personalized therapies.

methodsIn this study, fibroblast cells were isolated from a CSA patient and stained, determined by flow cytometry, and detected by immunocytochemistry. Subsequently, transfection optimization was performed with Lipofectamine.

resultsAfter 24 h of transfection with Cas9/sgRNA3 expression plasmid and single-stranded oligodeoxynucleotide (ssODN) donor, a transfection efficiency of 49.64% was achieved. The presence of the ssODN donor in the target gene region was examined by PCR-RFLP and PAGE, observing two fragments with approximate sizes of 116 and 161 bp. These results are consistent with insertion of the ssODN donor into the target gene region in fibroblast cells isolated from the patient.

conclusionsThe findings of this study indicate that the use of the CRISPR/Cas9 system with the designed oligo donor, under the tested conditions, has the potential to produce the expected changes at the target gene locus in patient fibroblast cells. Complementary studies using sequencing, along with examining different transfection conditions, could help more accurately confirm the findings of this study and further optimize this approach for future studies.