Document Type : Original Article
Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Department of Tissue Engineering and Applied Cell Science, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Objective: Beta-thalassemia is a group of inherited hematologic. The most HBB gene variant among Iranian
beta-thalassemia patients is related to two mutations of IVSII-1 (G>A) and IVSI-5 (G>C). Therefore, our aim of
this study is to use the knock in capability of CRISPR Cas9 system to investigate the correction of IVSII-1 (G>A)
variant in Iran.
Materials and Methods: In this experimental study, following bioinformatics studies, the vector containing
Puromycin resistant gene (PX459) was cloned individually by designed RNA-guided nucleases (gRNAs), and
cloning was confirmed by sequencing. Proliferation of TLS-12 was done. Then, the transfect was set up by the
vector with GFP marker (PX458). The PX459 vectors carrying the designed gRNAs together with Single-stranded
oligodeoxynucleotides (ssODNs) as healthy DNA pattern were transfected into TLS-12 cells. After taking the single
cell clones, molecular evaluations were performed on single clones. Sanger sequencing was then performed to
investigate homology directed repair (HDR).
Results: The sequencing results confirmed that all three gRNAs were successfully cloned into PX459 vector. In the
transfection phase, The TLS-12 containing PX459-gRNA/ssODN was selected. Molecular evaluations showed that
the HBB gene was cleaved by the CRISPR/Cas9 system, that indicates that the performance of non-homologous end
joining (NHEJ) repair system. Sequencing in some clones cleaved by the T7E1 enzyme showed that HDR was not
confirmed in these clones.
Conclusion: IVS-II-1 (G> A) mutation, which is the most common thalassemia mutation especially in Iran, the CRISPR/
Cas9 system was able to specifically target the HBB gene sequence. This could even lead to a correction in the
mutation and efficiency of the HDR repair system in future research.