Cellular reactivation of foetal haemoglobin transcription by the use of CRISPR/Cas9

Abstract

Sequential expression of embryonic, foetal and adult haemoglobin during human ontogeny has been a focus of numerous studies focusing on mechanisms of target gene switching in order to increase expression of foetal haemoglobin and consequently leading to significant improvement of clinical manifestation of haemoglobinopathies that include sickle cell disease and β-thalassaemia. Understanding the natural switch control from foetal to adult haemoglobin and actions of genes involved could lead to potentially lifesaving therapeutic approach. EKLF also known as KLF1 is an important regulatory protein involved in the γ- to β-globin gene switching mechanism that directly induces the expression of the β-globin gene and conversely represses γ-globin. In this study I sought to test a number of CRISPR/Cas9 systems with the purpose to disrupt the functions of KLF1, LRF and BCL11A genes and inhibit the γ- to β-globin gene switching in K562 cells. Relative quantification was performed for the assessment of all globin including γ globin expression as well as the target genes KLF1, BCL11A and LRF. The BCL11A quantification was not successful due to low expression of the same gene in K562 cells and was therefore not pursued. The levels of γ-globin mRNA varied between 48hrs and 96hrs post differentiation with hemin induction, showing a 1.97-fold induction for KLF1 knockdown by CRISPR/Cas9 as opposed to just after 48hrs and also when compared to untreated cells. The findings obtained in this dissertation support the induction of γ-globin expression upon targeting KLF1 through CRISPR/Cas9 mediated silencing. As a result, the effect of KLF1 inhibitory influence on γ-globin gene expression is removed. The same was not observed upon targeting LRF by CRISPR/Cas9, in which γ-globin remained low. Application of CRISPR technology in adult erythroid progenitors may provide a method for activating foetal haemoglobin expression in individuals with β-thalassaemia or sickle cell disease.