Efficient CRISPR-Cas9-based genome editing of β-globin gene on erythroid cells from homozygous β39-thalassemia patients.

Gene editing by the CRISPR-Cas9 nuclease system technology can be considered among the most promising strategies to correct hereditary mutations in a variety of monogenic diseases. In this paper, we present for the first time the correction, by CRISPR-Cas9 gene editing, of the β39-thalassemia mutation, one of the most frequent in the Mediterranean area. The results obtained demonstrated the presence of normal β-globin genes after CRISPR-Cas9 correction of the β39-thalassemia mutation performed on erythroid precursor cells from homozygous β39-thalassemia patients. This was demonstrated by allele-specific PCR and sequencing. Accumulation of corrected β-globin mRNA and relevant "" production of β-globin and adult hemoglobin (HbA) were found with high efficiency. The CRISPR-Cas9-forced HbA production levels were associated with a significant reduction of the excess of free α-globin chains. Genomic toxicity of the editing procedure (low indels and no off-targeting) was analyzed. The protocol might be the starting point for the development of an efficient editing of CD34 cells derived from β39 patients and for the design of combined treatments using, together with the CRISPR-Cas9 editing of the β-globin gene, other therapeutic approaches, such as, for instance, induction of HbA and/or fetal hemoglobin (HbF) using chemical inducers.