CRISPR/Cas9 Gene Editing of RYR2 in Human iPSC-Derived Cardiomyocytes to Probe Ca2+ Signaling Aberrancies of CPVT Arrhythmogenesis.
CRISPR/Cas9 Gene Editing of RYR2 in Human iPSC-Derived Cardiomyocytes to Probe Ca2+ Signaling Aberrancies of CPVT Arrhythmogenesis.
复制标题
对人 iPSC 来源的心肌细胞中的 RYR2 进行 CRISPR/Cas9 基因编辑，以探测 CPVT 心律失常发生的 Ca2 信号传导异常。
DOI：
10.1007/978-1-0716-2707-5_4
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Morad,Martin
中科院分区：
文献类型：
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作者：
Yamaguchi,Naohiro;Zhang,Xiao-Hua;Morad,Martin
人类诱导的多能干细胞（hiPSC）为研究心律失常相关基因突变的病理生理学的生物物理和分子机制提供了一个强大的平台。人iPSC可以通过正常或患病个体的真皮成纤维细胞的重编程产生，并分化成心肌细胞。从患有引起心律失常的点突变的患者身上获得活检通常是一个繁琐的过程，即使患者是可用的。然而，CRISPR/Cas9基因编辑系统的最新发展使得有可能在相对短的时间内在野生型hiPSC的所需基因座处引入与疟疾相关的点突变。我们利用该平台比较了心脏钙释放通道2型兰尼碱受体（RyR 2）基因点突变的心肌细胞的钙信号表型，因为超过200个RYR 2基因错义突变似乎与儿茶酚胺能多态性室性心动过速（CPVT 1）相关。我们已经创建了在RyR 2的不同结构域中携带突变的心肌细胞，不仅研究它们的Ca 2+信号传导结果，还研究它们与CPVT 1病理学相关的药物和结构域特异性。在本章中，我们描述了我们建立CRISPR/Cas9基因编辑的hiPSC衍生心肌细胞的程序。
Human-induced pluripotent stem cells (hiPSCs) provide a powerful platform to study biophysical and molecular mechanisms underlying the pathophysiology of genetic mutations associated with cardiac arrhythmia. Human iPSCs can be generated by reprograming of dermal fibroblasts of normal or diseased individuals and be differentiated into cardiac myocytes. Obtaining biopsies from patients afflicted with point mutations causing arrhythmia is often a cumbersome process even when patients are available. Recent development of CRISPR/Cas9 gene editing system makes it, however, possible to introduce arrhythmia-associated point mutations at the desired loci of the wild-type hiPSCs in relatively short times. This platform was used by us to compare the Ca2+signaling phenotypes of cardiomyocytes harboring point mutations in cardiac Ca2+release channel, type-2 ryanodine receptor (RyR2), since over 200 missense mutations inRYR2gene appear to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1). We have created cardiac myocytes harboring mutations in different domains of RyR2, to study not only their Ca2+signaling consequences but also their drug and domain specificity as related to CPVT1 pathology. In this chapter, we describe our procedures to establish CRISPR/Cas9 gene-edited hiPSC-derived cardiomyocytes.