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

• Published in: Gene Vol. 734; p. 144398
• Main Authors: Kalkan, Batuhan Mert, Kala, Ezgi Yagmur, Yuce, Melek, Karadag Alpaslan, Medine, Kocabas, Fatih
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.04.2020
• Subjects: Anemia; Anemia, Sickle Cell - genetics; beta-Globins - genetics; CRIPSR/Cas9; CRISPR-Cas Systems; DNA Breaks, Double-Stranded; DNA Repair; Gene editing; Gene Editing - methods; Genetic Vectors; HEK293 Cells; Humans; Mutation; RNA, Guide, CRISPR-Cas Systems; Sickle cell disease; Thalassemia; mM; DMSO; Gene editing; HBB; PKc; GVHD; HR; M; BSA; DMEM; ESC; FBS; HDR; µl; Sickle cell disease; crRNA; ml; DSBs; CRISPR; Anemia; HSPCs; Thalassemia; Cas; SCD; sgRNA; CRIPSR/Cas9; NHEJ; PAM
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2020.144398

[Display omitted] •HBB gene were targeted by spCas9 in close proximity to the SCD mutation.•SCD specific long gRNA exhibits indistinguishable cleavage activity on target locus.•Functional HBB HDR repair templates covered all known mutations in the HBB gene.•PAM sequence was replaced with HindIII site to assess HDR efficiency.•HDR template: Cas9-GFP vector 2:1 ratio yielded the highest HDR events/GFP+ cells. 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 β-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.