Genome editing the human beta globin locus for foetal haemoglobin production

Abstract

Amelioration of the symptoms of sickle cell disease or β-thalassaemia patients can be obtained by reactivation of the developmental program of foetal haemoglobin (HbF). This results in a raised level of foetal haemoglobin that can only be achieved in the clinical setting by firstly fully understanding the genetic switch from foetal (γ-globin) to adult haemoglobin (β-globin). Substantial progress has recently been made in this field, however the exact mechanism remains elusive. The combination of clinical research coupled with basic research should help in this regard. In a previous research conducted by a Borg et al (2010), a Maltese family was found to exhibit a nonsense variant, p. K288X, in KLF1 resulting in its partial loss of function, and elevation of HbF due to loss of repression by this gene. However, the heterogeneous distribution of HbF in the different family members with KLF1 haploinsufficiency, with a range of 3.3 to 20% HbF and in a collection of patients with borderline HbA2/HbF could not be fully explained by this gene on its own and it was concluded that other genetic elements were at play. A number of candidate genes were identified by Borg et al. following RNA-seq and ATAC-seq studies on the promoter region of KLF1. From this list, that included LRF, KLF1 and BCL11A, knock down studies were performed utilizing CRISPR/Cas9 technology to quantify transcripts of the human β-globin locus to determine the important cis- and trans- regions responsible for gamma globin gene (HBG1/2) induction and HbF production using various methods including Real Time PCR (qPCR) and to further understand the role of the targeted genes in regulation of variable HbF and in the switching mechanism from foetal to adult haemoglobin.