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Information Journal Paper

Title

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

Author(s)

 Servatian Nazli | ABROUN SAEID | Shahzadeh Fazeli Seyed Abolhassan | SOLEIMANI MASOUD | Issue Writer Certificate 

Pages

  176-183

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.

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    APA: Copy

    Servatian, Nazli, ABROUN, SAEID, & Shahzadeh Fazeli, Seyed Abolhassan. (2023). Investigating The Correction of IVS II-1 (G> A) Mutation in HBB Gene in TLS-12 Cell Line Using CRISPR/Cas9 System. CELL JOURNAL (YAKHTEH), 25(3), 176-183. SID. https://sid.ir/paper/1038190/en

    Vancouver: Copy

    Servatian Nazli, ABROUN SAEID, Shahzadeh Fazeli Seyed Abolhassan. Investigating The Correction of IVS II-1 (G> A) Mutation in HBB Gene in TLS-12 Cell Line Using CRISPR/Cas9 System. CELL JOURNAL (YAKHTEH)[Internet]. 2023;25(3):176-183. Available from: https://sid.ir/paper/1038190/en

    IEEE: Copy

    Nazli Servatian, SAEID ABROUN, and Seyed Abolhassan Shahzadeh Fazeli, “Investigating The Correction of IVS II-1 (G> A) Mutation in HBB Gene in TLS-12 Cell Line Using CRISPR/Cas9 System,” CELL JOURNAL (YAKHTEH), vol. 25, no. 3, pp. 176–183, 2023, [Online]. Available: https://sid.ir/paper/1038190/en

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