Highly efficient ESC genome editing with CRISPR/Cas۹ for production of laboratory models

Background: Beta-thalassemia is a group of hereditary blood disorders caused by mutations in the β-globin gene cluster resulting in variable phenotypes ranging from severe anemia to clinically asymptomatic individuals. This study aimed to produce an in vitro model of β-thalassemia using CRISPR/Cas۹ as an easily programmable, fast, more powerful, and efficient technique. Materials and Methods: Guide RNA (gRNA)-Cas۹ co-expression vectors were used for embryonic stem (ES) cell nucleofection. PCR, T۷EI, and Hbb-b۱ gene sequencing tests were done on extracted DNA to evaluate gene mutation. Following erythroid differentiation of ES cells, analysis of hemoglobin genes and erythroid transcription factors were assessed using a quantitative reverse transcription-polymerase chain reaction. Results: Sequencing data associated with clone ۳۱ confirmed the deletion of ۸۵۱ nucleotides between exon ۲ and ۳ in an Hbb-b۱ allele in this clone and Indel mutation in exon ۲ (-۴۰bp/+۳۸bp) from another allele of Hbb-b۱. Significant expression of erythroid transcription factors was observed in wild type, Hbb-b۱+/- and Hbb-b۱-/- groups. The hbb-b۱ gene expression in the Hbb-b۱+/- group significantly decreased, although the Hbb-b۱-/- group had zero expression. Conclusion: Utilizing an efficient erythroid differentiation method on the CRISPR/Cas۹-mediated Hbb-b۱ knock-out in ES cells provides accessibility to the laboratory thalassemia model. This method could be used to produce a mouse model of β-thalassemia intermedia (Hbbth۱/th۱ mice), which are required for the identification of the molecular basis of β-thalassemia and enable testing of the therapeutic approaches such as the recovery of functional β or γ hemoglobin chain.

Keywords:

Beta‐thalassemia , CRISPR‐Cas۹ system , Hbb‐b۱ , Mouse embryonic stem cell