Highly efficient ESC genome editing with CRISPR/Cas9 for production of laboratory models

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/Cas9 as an easily programmable, fast, more powerful, and efficient technique.

Guide RNA (gRNA)-Cas9 co-expression vectors were used for embryonic stem (ES) cell nucleofection. PCR, T7EI, and Hbb-b1 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.

Sequencing data associated with clone 31 confirmed the deletion of 851 nucleotides between exon 2 and 3 in an Hbb-b1 allele in this clone and Indel mutation in exon 2 (-40bp/+38bp) from another allele of Hbb-b1. Significant expression of erythroid transcription factors was observed in wild type, Hbb-b1+/- and Hbb-b1-/- groups. The hbb-b1 gene expression in the Hbb-b1+/- group significantly decreased, although the Hbb-b1-/- group had zero expression.

Utilizing an efficient erythroid differentiation method on the CRISPR/Cas9-mediated Hbb-b1 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 (Hbbth1/th1 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.