Investigating The Correction of IVS II-1 (G> A) Mutation in HBB Gene in TLS-12 Cell Line Using CRISPR/Cas9 System

Document Type : Original Article

Authors

1 Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

3 Department of Tissue Engineering and Applied Cell Science, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Abstract

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.

Keywords

References

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