SLC25A38 gene modification mediated by CRISPR/Cas9 in HEK293T cell line.

Sideroblastic anemia is a rare disease and one of the heterogeneous bone marrow disorders. Gene therapy is considered as a method for treating these patients. The CRISPR/Cas9 system can recognize specific genomic DNA sequences and create Double-Strand Breaks (DSBs) in DNA. In this study, we attempted to investigate the effect of different sgRNAs in creating targeted cleavage in the desired region using the CRISPR/Cas9 system and inducing changes in the disease-related gene. Following the design of six sgRNAs, recombinant pX458 and pX459 plasmids harboring the respective sgRNA sequences were constructed and transfected into HEK293T cells. After transfection, the efficiency of targeted cleavage of the designed sgRNAs was evaluated using polyacrylamide gel electrophoresis (PAGE)-based analysis of the target amplicons and subsequently confirmed by Sanger sequencing. Based on the sequencing results, it was shown that in three out of four samples examined, the CRISPR/Cas9 system successfully created targeted disruption at the desired site. Here, we report that the choice of sgRNA target site affects its targeted cleavage activity and show that the combination of guideRNA with SpCas9 can effectively delete the exonic fragment of the SLC25A38 gene in HEK293T cells.