title: CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation.
abstract: Mutations in SCN1A, the gene encoding the α subunit of Nav1.1 channel, can cause epilepsies with wide ranges of clinical phenotypes, which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project, CRISPR/Cas9- and TALEN-mediated genome-editing techniques were applied to induced pluripotent stem cell (iPSC)-based-disease model to explore the mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9, we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G, which led to no current of Nav1.1 in exogenously transfected system, influenced the properties of not only Nav current amount, but also Nav activation in Nav1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons, and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in the patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly, when the frequencies of both sIPSCs and sEPSCs were further analyzed, we found the whole postsynaptic activity transferred from the inhibition-dominated state to excitation in patient-derived neuronal networks, suggesting that changes in sIPSCs alone were sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity. In summary, our findings fill the gap of our knowledge regarding the relationship between SCN1A mutation effect recorded on exogenously transfected cells and on Nav1.1-expressing neurons, and reveal the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation.
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, China.|Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: C
authors.initials: C
authors.lastname: Gao
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: W
authors.initials: W
authors.lastname: Chen
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: W
authors.initials: W
authors.lastname: Ma
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Collaborative Innovation Center for Neurogenetics and Channelopathies, Institute of Neuroscience, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Long
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: H
authors.initials: H
authors.lastname: Xu
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: X
authors.initials: X
authors.lastname: Guo
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Sciences, Central South University, Changsha, China.
authors.forename: S
authors.initials: S
authors.lastname: Deng
authors.affiliation: Department of Anatomy and Neurobiology, School of Basic Medical Sciences, Central South University, Changsha, China.
authors.forename: X
authors.initials: X
authors.lastname: Yan
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: D
authors.initials: D
authors.lastname: Yu
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: G
authors.initials: G
authors.lastname: Pan
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: L
authors.initials: L
authors.lastname: Lai
authors.affiliation: Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Collaborative Innovation Center for Neurogenetics and Channelopathies, Institute of Neuroscience, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
authors.forename: W
authors.initials: W
authors.lastname: Liao
authors.affiliation: Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.|Department of Anatomy and Neurobiology, School of Basic Medical Sciences, Central South University, Changsha, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Li
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002648:Child; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D055724:Electrophysiological Phenomena; D004827:Epilepsy / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D009154:Mutation / Q000235:genetics*; D062550:NAV1.1 Voltage-Gated Sodium Channel / Q000235:genetics* / Q000378:metabolism; D009415:Nerve Net / Q000378:metabolism*
chemical_list: D062550:NAV1.1 Voltage-Gated Sodium Channel; C568242:SCN1A protein, human