人工诱导多能干细胞（hiPSCs）已广泛应用于心脏疾病模型、药物发现和再生医学领域，因为它们可以分化为特定患者的心肌细胞。长QT综合征3型（LQT3）是较为恶性的先天性长QT综合征（LQTS）变体之一，具有对钠通道的SCN5A增产作用。此外，LQTS基因中主要的致病变异是单核苷酸替换（错义）和小插入/缺失（INDEL）。CRISPR/Cas9基因组编辑已被用于创建等基因背景的等基因多能干细胞，以控制单核苷酸变异的致病性。在本研究中，我们描述了一个优化和快速的方案，利用核糖核酸蛋白（RNP）和单链寡核苷酸（ssODN）将杂合性LQT3特异变异引入健康对照hIPSCs中。根据该方案，我们成功筛选出带有杂合性LQT3致病变异（SCN5A±）的hiPSCs，筛选效率高（69个样本中的6个），并确认无离靶效应，正常核型，高碱性磷酸酶活性，未受到多能性的影响，并在2周内形成了体外胚胎体结构。此外，我们还提供了将hiPSCs成功分化为心肌细胞，并利用多电极阵列评估电生理特性的方案。该方案也适用于将其他特定疾病变异引入和/或纠正hiPSCs，以进行未来的药物筛选和基因治疗开发。©2023.作者。

Human induced pluripotent stem cells (hiPSCs) have been widely used in cardiac disease modelling, drug discovery, and regenerative medicine as they can be differentiated into patient-specific cardiomyocytes. Long QT syndrome type 3 (LQT3) is one of the more malignant congenital long QT syndrome (LQTS) variants with an SCN5A gain-of-function effect on the gated sodium channel. Moreover, the predominant pathogenic variants in LQTS genes are single nucleotide substitutions (missense) and small insertion/deletions (INDEL). CRISPR/Cas9 genome editing has been utilised to create isogenic hiPSCs to control for an identical genetic background and to isolate the pathogenicity of a single nucleotide change. In this study, we described an optimized and rapid protocol to introduce a heterozygous LQT3-specific variant into healthy control hiPSCs using ribonucleoprotein (RNP) and single-stranded oligonucleotide (ssODN). Based on this protocol, we successfully screened hiPSCs carrying a heterozygous LQT3 pathogenic variant (SCN5A±) with high efficiency (6 out of 69) and confirmed no off-target effect, normal karyotype, high alkaline phosphatase activity, unaffected pluripotency, and in vitro embryonic body formation capacity within 2 weeks. In addition, we also provide protocols to robustly differentiate hiPSCs into cardiomyocytes and evaluate the electrophysiological characteristics using Multi-electrode Array. This protocol is also applicable to introduce and/or correct other disease-specific variants into hiPSCs for future pharmacological screening and gene therapeutic development.© 2023. The Author(s).