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


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


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.


  1. Akhavan-Niaki H, Derakhshandeh-Peykar P, Banihashemi A, Mostafazadeh A, Asghari B, Ahmadifard MR, et al. A comprehensive molecular characterization of beta thalassemia in a highly heterogeneous population. Blood Cells Mol Dis. 2011; 47(1): 29-32.
  2. Amin SS, Jalal SD, Ali KM, Mohammed AI, Rasool LK, Osman TJ. Beta-thalassemia intermedia: a single thalassemia center experience from northeastern Iraq. Biomed Res Int. 2020; 2020: 2807120.
  3. Rahimi Z, Muniz A, Akramipour R, Tofieghzadeh F, Mozafari H, Vaisi- Raygani A, et al. Haplotype analysis of beta thalassemia patients in Western Iran. Blood Cells Mol Dis. 2009; 42(2): 140-143.
  4. Abolghasemi H, Amid A, Zeinali S, Radfar MH, Eshghi P, Rahiminejad MS, et al. Thalassemia in Iran: epidemiology, prevention, and management. J Pediatr Hematol Oncol. 2007; 29(4): 233-238.
  5. D’Andrea G, Di Perna P, Brancaccio V, Faioni EM, Castaman G, Cibelli G, et al. A novel G-to-A mutation in the intron-N of the protein S gene leading to abnormal RNA splicing in a patient with protein S deficiency. Haematologica. 2003; 88(4): 459-464.
  6. Anurathapan U, Hongeng S, Pakakasama S, Songdej D, Sirachainan N, Pongphitcha P, et al. Hematopoietic stem cell transplantation for severe thalassemia patients from haploidentical donors using a novel conditioning regimen. Biol Blood Marrow Transplant. 2020; 26(6): 1106-1112.
  7. Sadelain M, Boulad F, Galanello R, Giardina P, Locatelli F, Maggio A, et al. Therapeutic options for patients with severe beta-thalassemia: the need for globin gene therapy. Hum Gene Ther. 2007; 18(1): 1-9.
  8. Sadelain M, Lisowski L, Samakoglu S, Rivella S, May C, Riviere I. Progress toward the genetic treatment of the beta-thalassemias. Ann N Y Acad Sci. 2005; 1054: 78-91.
  9. Mansilla-Soto J, Riviere I, Boulad F, Sadelain M. Cell and gene therapy for the beta-thalassemias: advances and prospects. Hum Gene Ther. 2016; 27(4): 295-304.
  10. Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. 2021; 6(1): 53.
  11. Foss DV, Hochstrasser ML, Wilson RC. Clinical applications of CRISPR-based genome editing and diagnostics. Transfusion. 2019; 59(4): 1389-1399.
  12. Xu X, Wan T, Xin H, Li D, Pan H, Wu J, et al. Delivery of CRISPR/ Cas9 for therapeutic genome editing. J Gene Med. 2019; 21(7): e3107.
  13. Li B, Niu Y, Ji W, Dong Y. Strategies for the CRISPR-Based Therapeutics. Trends Pharmacol Sci. 2020; 41(1): 55-65.
  14. Mani I. CRISPR-Cas9 for treating hereditary diseases. Prog MolBiol Transl Sci. 2021; 181: 165-183.
  15. Jiang F, Doudna JA. CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biophys. 2017; 46: 505-529.
  16. Xue C, Greene EC. DNA repair pathway choices in CRISPR-Cas9- mediated genome editing. Trends Genet. 2021; 37(7): 639-656.
  17. Khadempar S, Familghadakchi S, Motlagh RA, Farahani N, Dashtiahangar M, Rezaei H, et al. CRISPR-Cas9 in genome editing: its function and medical applications. J Cell Physiol. 2019; 234(5): 5751-5761.
  18. Zaboikin M, Zaboikina T, Freter C, Srinivasakumar N. Non-homologous end joining and homology directed dna repair frequency of double-stranded breaks introduced by genome editing reagents. PLoS One. 2017; 12(1): e0169931.
  19. Sansbury BM, Hewes AM, Kmiec EB. Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair. Commun Biol. 2019; 2: 458.
  20. Hewes AM, Sansbury BM, Kmiec EB. The diversity of genetic outcomes from CRISPR/Cas gene editing is regulated by the length of the symmetrical donor DNA template. Genes (Basel). 2020; 11(10): 1160.
  21. Sansbury BM, Hewes AM, Tharp OM, Masciarelli SB, Kaouser S, Kmiec EB. Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas. Sci Rep. 2022; 12(1): 8132.
  22. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012; 13: 134.
  23. Giacalone JC, Sharma TP, Burnight ER, Fingert JF, Mullins RF, Stone EM, et al. CRISPR-Cas9-based genome editing of human induced pluripotent stem cells. Curr Protoc Stem Cell Biol. 2018; 44: 5B.7.1-5B.7.22.
  24. Angastiniotis M, Petrou M, Loukopoulos D, Modell B, Farmakis D, Englezos P, et al. The prevention of thalassemia revisited: a historical and ethical perspective by the thalassemia international federation. Hemoglobin. 2021; 45(1): 5-12.
  25. Poggiali E, Cassinerio E, Zanaboni L, Cappellini MD. An update on iron chelation therapy. Blood Transfus. 2012; 10(4): 411-422.
  26. Gluckman E, Cappelli B, Bernaudin F, Labopin M, Volt F, Carreras J, et al. Sickle cell disease: an international survey of results of HLA-identical sibling hematopoietic stem cell transplantation. Blood. 2017; 129(11): 1548-1556.
  27. Mahdieh N, Rabbani B. Beta thalassemia in 31,734 cases with HBB gene mutations: Pathogenic and structural analysis of the common mutations; Iran as the crossroads of the Middle East. Blood Rev. 2016; 30(6): 493-508.
  28. Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, et al. CRISPR/ Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015; 6(5): 363-372.
  29. Wattanapanitch M, Damkham N, Potirat P, Trakarnsanga K, Janan M, U-Pratya Y, et al. One-step genetic correction of hemoglobin E/ beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system. Stem Cell Res Ther. 2018; 9(1): 46.
  30. Gabr H, El Ghamrawy MK, Almaeen AH, Abdelhafiz AS, Hassan AOS, El Sissy MH. CRISPR-mediated gene modification of hematopoietic stem cells with beta-thalassemia IVS-1-110 mutation. Stem Cell Res Ther. 2020; 11(1): 390.