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，然而，将已知致病突变引入hPSCs的过程既耗时又费力。基编辑是一种新兴技术，它能够轻松地将点突变引入活细胞基因组中的特定位点。在本研究中，我们设计了一种集成的环状质粒载体，该载体表达单个引导RNA（sgRNA）并配备腺嘌呤基编辑器（ABE）或胞嘧啶基编辑器（CBE）。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综合征的疾病特异性突变引入hPSCs，以模拟LQT1、LQT2和LQT3。利用多电极阵列（MEAs）对hPSC来源的心肌细胞（hPSC-CMs）进行电生理分析，发现经过编辑的hPSC-CMs动作电位持续时间显著增加。最后，我们将新型的布鲁加达综合征相关突变引入hPSCs，证明了这种突变可导致异常的电生理反应。本研究表明，环状质粒编码的基编辑器（epi-BEs）能够高效地生成针对特定疾病的hPSC模型。