Badat, M.Ejaz, A.Hua, P.Rice, S.Zhang, W.Hentges, L.D.Fisher, C.A.Denny, N.Schwessinger, R.Yasara, N.Roy, N.B.A.Issa, F.Roy, A.Telfer, P.Hughes, J.Mettananda, S.Higgs, D.R.Davies, J.O.J.2023-04-272023-04-272023Nature Communications.2023;14(1):2238.2041-1723http://repository.kln.ac.lk/handle/123456789/26124indexed in MEDLINE.Haemoglobin E (HbE) β-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE β-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe β-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (β-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.enβ-thalassaemiaHaemoglobinArticle