Abstract
β-thalassemia, caused by mutations in the human hemoglobin β (HBB) gene, is one of the most common genetic diseases in the world. The HBB −28(A>G) mutation is one of the five most common mutations in Chinese patients with β-thalassemia. However, few studies have been conducted to understand how this mutation affects the expression of pathogenesis-related genes, including globin genes, due to limited homozygote clinical materials. Therefore, we developed an efficient technique using CRISPR/Cas9 combined with asymmetric single-stranded oligodeoxynucleotides (assODNs) to generate a K562 cell model with HBB −28(A>G) named K562–28(A>G). Then, we systematically analyzed the differences between K562–28(A>G) and K562 at the transcriptome level by high-throughput RNA-seq before and after erythroid differentiation. We found that the HBB −28(A>G) mutation not only disturbed the transcription of HBB, but also decreased the expression of HBG, which may further aggravate the thalassemia phenotype and partially explain the more severe clinical outcome of β-thalassemia patients with the HBB −28(A>G) mutation. Moreover, we found that the K562–28(A>G) cell line is more sensitive to hypoxia and shows a defective erythrogenic program compared with K562 before differentiation. Importantly, all abovementioned abnormalities in K562–28(A>G) were reversed after correction of this mutation with CRISPR/Cas9 and assODNs, confirming the specificity of these phenotypes. Overall, this is the first time to analyze the effects of the HBB −28(A>G) mutation at the whole-transcriptome level based on isogenic cell lines, providing a landscape for further investigation of the mechanism of β-thalassemia with the HBB −28(A>G) mutation.
Highlights
Reviewed by: Mo Li, King Abdullah University of Science and Technology, Saudi Arabia Ling Li, Beckman Research Institute, City of Hope, United States Liren Wang, East China Normal University, China
We found that HBB was transcriptionally prevented by the mutation, and the K562−28(A > G) cell line is more sensitive to hypoxia and present a defective erythrogenic program when compared with wild-type K562 before erythroid differentiation by Gene Ontology (GO) and KEGG analysis
That using single-stranded oligodeoxynucleotides (ssODNs) (Niu et al, 2016) or asymmetric doublestrand DNA (Paquet et al, 2016) as a repair template resulted in a higher efficiency of accurate replacement of target sequences through homology-directed repair (HDR), we developed a technology that combined CRISPR/Cas9 with asymmetric ssODNs
Summary
Reviewed by: Mo Li, King Abdullah University of Science and Technology, Saudi Arabia Ling Li, Beckman Research Institute, City of Hope, United States Liren Wang, East China Normal University, China. High-throughput RNA sequencing (RNAseq) was used to compare control samples with patient samples carrying a novel HBB mutation (HBB: c.51C > T) It shows that hemopoiesis, heme biosynthesis, response to oxidative stress, and other cellular activity pathways were directly or indirectly enriched by differentially expressed genes related to β-thalassemia (Taghavifar et al, 2019), suggesting that genomewide RNA-seq analysis is a useful approach to understand the mechanism of β-thalassemia with different mutations. A human umbilical cord blood–derived erythroid progenitor (HUDEP) cell line was established and used as a disease model because of its differentiation ability to produce enucleated red blood cells (Kurita et al, 2013; Traxler et al, 2016; Grevet et al, 2018) This cell line is derived from primary hematopoietic stem cells with E6/E7 immortalized basophilic erythroblasts, which depends on erythropoietin (EPO) and cytokine stem cell factor (SCF) for survival, so it is expensive to maintain and hard to do gene editing (Couch et al, 2019). We chose the K562 cell line to generate the thalassemia disease cell model for its ready availability, cost-effective maintenance, relatively mature differentiation conditions, and high efficiency of gene editing (Dean et al, 1983; Witt et al, 2000; Ma et al, 2013; DeWitt et al, 2016; Shariati et al, 2016; Ghosh et al, 2018)
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