Genetics & Genome Biology Program, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada; The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
Stem Cell and Cancer Research Institute, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton L8S 4L8, Canada.
Stem Cell Reports. 2018 Nov 13;11(5):1211-1225. doi: 10.1016/j.stemcr.2018.10.003. Epub 2018 Nov 1.
Autism spectrum disorder (ASD) is phenotypically and genetically heterogeneous. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of ten ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NGN2)-directed induction of iPSCs allowed production of excitatory neurons, and mutant proteins were not detectable. RNA sequencing revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory postsynaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2-, and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.
自闭症谱系障碍（ASD）在表型和遗传上具有异质性。我们提出了一种 CRISPR 基因编辑策略，用于插入蛋白标签和提前终止位点，从而创建一个诱导多能干细胞（iPSC）敲除资源，用于研究十个与 ASD 相关的基因（AFF2/FMR2、ANOS1、ASTN2、ATRX、CACNA1C、CHD8、DLGAP2、KCNQ2、SCN2A、TENM1）的功能。神经基因 2（NGN2）定向诱导 iPSC 允许产生兴奋性神经元，并且无法检测到突变蛋白。RNA 测序揭示了几个神经元网络的趋同。使用膜片钳和多电极阵列方法，不同突变测量的电生理缺陷是不同的。然而，它们最终导致突触活动的一致减少，包括 AFF2/FMR2、ASTN2、ATRX、KCNQ2 和 SCN2A 缺失神经元中自发兴奋性突触后电流频率的降低。尽管 ASD 易感基因属于不同的基因本体论，但同基因干细胞资源可以揭示常见的功能表型，例如功能连接减少。
