Data_Sheet_1_Base Editing Mediated Generation of Point Mutations Into Human Pluripotent Stem Cells for Modeling Disease.docx

Human pluripotent stem cells (hPSCs) are a powerful platform for disease modeling and drug discovery. However, the introduction of known pathogenic mutations into hPSCs is a time-consuming and labor-intensive process. Base editing is a newly developed technology that enables facile introduction of point mutations into specific loci within the genome of living cells. Here, we design an all-in-one episomal vector that expresses a single guide RNA (sgRNA) with an adenine base editor (ABE) or a cytosine base editor (CBE). Both ABE and CBE can efficiently introduce mutations into cells, A-to-G and C-to-T, respectively. We introduce disease-specific mutations of long QT syndrome into hPSCs to model LQT1, LQT2, and LQT3. Electrophysiological analysis of hPSC-derived cardiomyocytes (hPSC-CMs) using multi-electrode arrays (MEAs) reveals that edited hPSC-CMs display significant increases in duration of the action potential. Finally, we introduce the novel Brugada syndrome-associated mutation into hPSCs, demonstrating that this mutation can cause abnormal electrophysiology. Our study demonstrates that episomal encoded base editors (epi-BEs) can efficiently generate mutation-specific disease hPSC models.

人类多能干细胞（human pluripotent stem cells, hPSCs）是用于疾病建模与药物研发的强大研究平台。然而，将已知致病突变引入人类多能干细胞的操作耗时费力。碱基编辑是一项新兴技术，可便捷地将点突变引入活细胞基因组的特定位点。本研究设计了一种一体化游离型载体，可同时表达单引导RNA（single guide RNA, sgRNA）以及腺嘌呤碱基编辑器（adenine base editor, ABE）或胞嘧啶碱基编辑器（cytosine base editor, CBE）。腺嘌呤碱基编辑器与胞嘧啶碱基编辑器均可高效介导细胞内的突变引入，分别实现A→G与C→T的碱基替换。本研究将长QT综合征（long QT syndrome）的疾病特异性突变引入人类多能干细胞，以构建LQT1、LQT2及LQT3型疾病模型。采用多电极阵列（multi-electrode arrays, MEAs）对人类多能干细胞诱导的心肌细胞（human pluripotent stem cell-derived cardiomyocytes, hPSC-CMs）进行电生理分析，结果显示编辑后的心肌细胞动作电位时程显著延长。最后，本研究将一种新型布鲁加达综合征（Brugada syndrome）相关突变引入人类多能干细胞，证实该突变可导致异常电生理表型。本研究证实，游离型载体编码的碱基编辑器（episomal encoded base editors, epi-BEs）可高效构建具有突变特异性的疾病相关人类多能干细胞模型。