Impairment of F8 Gene Function in vitro with CRISPR-Cas9 Technology
Project no. 20393 · Status: Kapanmış (Yayın bekleniyor) · 2019
Summary
<p>Hemophilia A is a rare hereditary disease that develops due to a lack of the F8. Intron 1 and intron 22 inversions, point mutations, deletions, insertions and rearrangements mutations, occurring in F8, cause hemophilia A disease. Approximately half of the cases with severe hemophilia A, or about %30 of all cases, have "F8 intron 22 inversion mutation". Point mutations (single nucleotide changes) are considered to be the most common genetic defects. Hemophilia is a single gene disease that making it suitable candidate for genetic therapy.</p><p>Correction of mutations in the F8 gene with gene editing technologies may be a critical approach in the treatment of hemophilia. In this study, the creation of a Hemophilia A cell model by cutting double-stranded DNA from previously determined regions of the F8 gene and creating an indel (Inversion-Deletion) mutation with CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology in the Hepatic Stellate cell (LX-2) line has been researched. Four different sgRNAs designed for this purpose were subjected to efficiency testing and determined to have high efficiency, then they were ligated into plasmid vectors and cloned. It was determined that different sgRNAs were properly attached to the vectors during the cloning phase by DNA sequencing using the Sanger method. F8 gene and factor VIII protein expression were checked and detected by expression test and Western blot method in target cells (LX-2) before transfection, respectively. In addition, factor VIII protein activity assay was performed by Chromogenic method in Liver Stellate (LX-2), THLE-2, HEK 293T and HEPG2 cells. Plasmid vectors containing purified gRNA and Cas9 were transfected into nanovesicle producer HEK-293T cells. It was determined that nano vesicles containing 4 different target-specific sgRNA complexes were produced in HEK-293T cells and transfection took place. The resulting CRISPR Cas9 system was successfully transfected into LX-2 healthy liver Stellate cells (target cells). The red fluorescent signal originating from the CherryPicker fluorescent marker on the nano-vesicle surface was detected under the microscope in LX-2 cells and it was shown that the transfection was successful. Gene editing was confirmed by Sanger sequencing.</p><p>In conclusion, the construction of hemophilia cell model was achieved using CRISPR Cas9 technology . In the future, it is predicted that diagnosis, follow-up and treatment approaches in hemophilia can be investigated in-vitro using this cell model.</p>