GTAC enables parallel genotyping of multiple genomic loci with chromatin accessibility profiling in single cells

Understanding clonal evolution and cancer development requires experimental approaches for characterizing the consequences of somatic mutations on gene regulation. However, no methods currently exist that efficiently link high-content chromatin accessibility with high-confidence genotyping in single cells. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), enabling accurate mutation detection at multiple amplified loci, coupled with robust chromatin accessibility readout. We applied GTAC to primary acute myeloid leukemia, obtaining high-quality chromatin accessibility profiles and clonal identities for multiple mutations in 88% of cells. We traced chromatin variation throughout clonal evolution, showing the restriction of different clones to distinct differentiation stages. Furthermore, we identified switches in transcription factor motif accessibility associated with a specific combination of driver mutations, which biased transformed progenitors toward a leukemia stem cell-like chromatin state. GTAC is a powerful tool to study clonal heterogeneity across a wide spectrum of pre-malignant and neoplastic conditions

Direct correction of haemoglobin E β-thalassaemia using base editors

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