Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC)

Abstract

Objectives: Duchene muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations in the DMD gene, resulting in the absence of dystrophin expression leading to membrane fragility and myofibril necrosis in the muscle cells. Because of progressive weakness in the skeletal and cardiac muscles, premature death is inevitable. There is no curative treatment available for DMD. In recent years, advances in genetic engineering tools have made it possible to manipulate gene sequences and accurately modify disease-causing mutations. CRISPR/Cas9 technology is a promising tool for gene editing because of its ability to induce double-strand breaks in the DNA.

Materials and methods: In this study for the exon-skipping approach, we designed a new pair of guide RNAs (gRNA) to induce large deletion of exons 48 to 53 in the DMD gene in the human skeletal muscle cell line (HSkMC), in order to correct the frame of the gene.

Results: Data showed successful editing of DMD gene by deletion of exons 48 to 53 and correction of the reading frame in edited cells. Despite a large deletion in the edited DMD gene, the data of real-time PCR, immune florescent staining demonstrated successful expression of truncated dystrophin in edited cells.

Conclusion: This study demonstrated that the removal of exons 48-53 by the CRISPR / Cas9 system did not alter the expression of the DMD gene due to the preservation of the reading frame of the gene.

Keywords: CRISPR/Cas9; DMD; Dystrophin; Gene editing; HSkMC.

Figure 1

Schematic construct map and primer site according to our design for edition of dystrophin

Figure 2

Detection of the edited cells by PCR. A) Amplification of 498bp in edited cells. M (DNA Marker 1kb, Cinnagen), C-(negative control or non-edited HEK-293 cells), 1-8 (edited HEK-293 cells). B) Negative control (1), w- HSkMC or no edited cells (2), b-HSkMC, cells infected with PX458 empty or backbone (3), Amplification of 498bp fragment in e-HSkMC or edited cells (4), and DNA marker 1kb (Cinnagen. Iran) (M)

Figure 3

Dystrophin expression in the edited cells (e-HSkMC) and non-edited cells (w-HSkMC). GAPDH was used as an internal control gene. A) Plot demonstrates the total RNA expression in edited (e-HSkMC) and non-edited cells (w-HSkMC) checked by primer designed for exons 15 and 16. B) Plot demonstrated deletion of exons 48-53 in the edited cell, so amplification was not seen by primer for exons 47 and 48 in edited cells (P-value >0.05)

Figure 4

According to analysis of the DMD gene in www.edystrophin.genouest.org, deletion of exons 48-53 resulted in removal of a part of R18 to R 22 that is located in the rod domain. Rod domain of dystrophin is composed of 24 repeats (R1 to R24) homologue to spectrin repeats and 4 hinges. Deletion of exons 48-55 modeling from repeat 17 to repeat 23 from I-Tasser ( score: -0.94). Blue: R17, Violet: R18, Cyan: R22, Green: R23

Figure 5

Immune fluorescent staining using dystrophin Ab and DAPI as counter-stain in edited cells (e-HSkMC) and non-edited cells (w-HSkMC). A) e-HSkMC. B) w-HSkMC. Both eHSkMC and wHSkMC showed a positive result in immune fluorescent staining using dystrophin Ab. These results demonstrate that dystrophin could express in the edited cells