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Stem Cell Reports. 2018 Nov 13;11(5):1211-1225. doi: 10.1016/j.stemcr.2018.10.003. Epub 2018 Nov 1.

Complete Disruption of Autism-Susceptibility Genes by Gene Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons.

Author information

1
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.
2
Stem Cell and Cancer Research Institute, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton L8S 4L8, Canada.
3
Developmental & Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
4
Developmental & Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3H7, Canada.
5
The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
6
Stem Cell and Cancer Research Institute, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton L8S 4L8, Canada. Electronic address: singhk2@mcmaster.ca.
7
Developmental & Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3H7, Canada. Electronic address: jellis@sickkids.ca.
8
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; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3H7, Canada; McLaughlin Centre, University of Toronto, Toronto, ON M5S 3H7, Canada. Electronic address: stephen.scherer@sickkids.ca.

Abstract

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.

KEYWORDS:

CRISPR; NGN2; StopTag; autism; convergence; iPSC; isogenic; knockout; sEPSC

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