Development of gene editing strategies for human beta-globin (HBB) gene mutations

Creative Commons License

Kalkan B. M., Kala E. Y., Yuce M., Karadağ Alpaslan M., KOCABAŞ F.

GENE, cilt.734, 2020 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 734
  • Basım Tarihi: 2020
  • Doi Numarası: 10.1016/j.gene.2020.144398
  • Dergi Adı: GENE
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Aquatic Science & Fisheries Abstracts (ASFA), Artic & Antarctic Regions, BIOSIS, CAB Abstracts, EMBASE, Food Science & Technology Abstracts, MEDLINE, Veterinary Science Database
  • Anahtar Kelimeler: Sickle cell disease, Thalassemia, Anemia, Gene editing, CRIPSR/Cas9, SICKLE-CELL-DISEASE, HEMATOPOIETIC STEM-CELLS, PRENATAL-DIAGNOSIS, HEMOGLOBINOPATHIES, TRANSPLANTATION, THALASSEMIA, METABOLISM, CHILDREN, REPAIR, ASSAY
  • Ondokuz Mayıs Üniversitesi Adresli: Evet

Özet

Recent developments in gene editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene editing tools are promising candidates for clinical applications, especially for treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in human beta-globin gene (HBB). Clinical strategies have demonstrated substantial success, however there is not any permanent cure for SCD available. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (gRNA) to induce sequence-specific DNA double strand break (DSB). When this accompanies a repair template, it allows repairing the mutated gene. In this study, it was aimed to target HBB gene via CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in human cell line, by Homology Directed Repair (HDR). We have achieved to induce target specific nucleotide changes on HBB gene in the locus of mutation causing SCD. The effect of on-target activity of bone fide standard gRNA and newly developed longer gRNA were examined. It is observed that longer gRNA has higher affinity to target DNA while having the same performance for targeting and Cas9 induced DSBs. HDR mechanism was triggered by co-delivery of donor DNA repair templates in circular plasmid form. In conclusion, we have suggested methodological pipeline for efficient targeting with higher affinity to target DNA and generating desired modifications on HBB gene.