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.

人源多能干细胞（hPSCs）作为疾病建模和药物发现的有效平台，具有显著优势。然而，将已知的致病突变引入hPSCs的过程既耗时又费力。基于编辑的基因编辑技术，使得在活细胞基因组中特定位点轻易引入点突变成为可能。本研究中，我们设计了一种集成的环状质粒载体，该载体表达单链引导RNA（sgRNA）并与腺嘌呤碱基编辑器（ABE）或胞嘧啶碱基编辑器（CBE）相结合。ABE和CBE均能高效地将突变引入细胞中，分别实现A到G和C到T的转换。我们将长QT综合征的特定致病突变引入hPSCs，以构建LQT1、LQT2和LQT3的疾病模型。利用多电极阵列（MEAs）对hPSC衍生的心肌细胞（hPSC-CMs）进行电生理分析表明，经过编辑的hPSC-CMs的动作电位持续时间显著增加。最终，我们还将新型Brugada综合征相关突变引入hPSCs，证实该突变可导致异常的电生理现象。本研究证实，环状质粒编码的碱基编辑器（epi-BEs）能够高效地生成针对特定突变的疾病hPSC模型。