PDF(Pubmed)
Abstract：
β-thalassemia/HbE results from mutations in the β-globin locus that impede the production of functional adult hemoglobin. Base editors (BEs) could facilitate the correction of the point mutations with minimal or no indel creation, but its efficiency and bystander editing for the correction of β-thalassemia mutations in coding and non-coding regions remains unexplored. Here, we screened BE variants in HUDEP-2 cells for their ability to correct a spectrum of β-thalassemia mutations that were integrated into the genome as fragments of HBB. The identified targets were introduced into their endogenous genomic location using BEs and Cas9/homology-directed repair (HDR) to create cellular models with β-thalassemia/HbE. These β-thalassemia/HbE models were then used to assess the efficiency of correction in the native locus and functional β-globin restoration. Most bystander edits produced near target sites did not interfere with adult hemoglobin expression and are not predicted to be pathogenic. Further, the effectiveness of BE was validated for the correction of the pathogenic HbE variant in severe β0/βE-thalassaemia patient cells. Overall, our study establishes a novel platform to screen and select optimal BE tools for therapeutic genome editing by demonstrating the precise, efficient, and scarless correction of pathogenic point mutations spanning multiple regions of HBB including the promoter, intron, and exons.
摘要：
β-地中海贫血/HbE是由β-珠蛋白基因座中的突变引起的，该突变阻碍了功能性成人血红蛋白的产生。碱基编辑器（BE）可以在最少或没有indel创建的情况下促进点突变的校正，但其在编码区和非编码区β-地中海贫血突变校正中的效率和旁观者编辑仍未被探索。这里,我们在HUDEP-2细胞中筛选了BE变异体纠正β-地中海贫血突变谱的能力,这些突变作为HBB片段整合到基因组中.使用BE和Cas9/同源性指导修复(HDR)将鉴定的靶标引入其内源性基因组位置,以创建具有β-地中海贫血/HbE的细胞模型。然后使用这些β-地中海贫血/HbE模型来评估天然基因座的校正效率和功能性β-珠蛋白恢复。大多数在靶位点附近产生的旁观者编辑不会干扰成人血红蛋白表达，也不会被预测为致病性。Further,在重度β0/βE地中海贫血患者细胞中,验证了BE纠正致病性HbE变异的有效性.总的来说,我们的研究建立了一个新颖的平台来筛选和选择用于治疗性基因组编辑的最佳BE工具，高效,以及跨越HBB的多个区域包括启动子的致病点突变的无疤痕校正，内含子,和外显子。
