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

Eric Deneault; Sean H White; Deivid C Rodrigues; P Joel Ross; Muhammad Faheem; Kirill Zaslavsky; Zhuozhi Wang; Roumiana Alexandrova; Giovanna Pellecchia; Wei Wei; Alina Piekna; Gaganjot Kaur; Jennifer L Howe; Vickie Kwan; Bhooma Thiruvahindrapuram; Susan Walker; Anath C Lionel; Peter Pasceri; Daniele Merico; Ryan K C Yuen; Karun K Singh; James Ellis; Stephen W Scherer

doi:10.1016/j.stemcr.2018.10.003

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

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

Authors

Eric Deneault¹, Sean H White², Deivid C Rodrigues³, P Joel Ross³, Muhammad Faheem¹, Kirill Zaslavsky⁴, Zhuozhi Wang⁵, Roumiana Alexandrova⁵, Giovanna Pellecchia⁵, Wei Wei³, Alina Piekna³, Gaganjot Kaur⁵, Jennifer L Howe⁵, Vickie Kwan², Bhooma Thiruvahindrapuram⁵, Susan Walker¹, Anath C Lionel¹, Peter Pasceri³, Daniele Merico⁵, Ryan K C Yuen¹, Karun K Singh⁶, James Ellis⁷, Stephen W Scherer⁸

Affiliations

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Autistic Disorder / genetics*
Autistic Disorder / physiopathology*
Cell Line
Electrodes
Gene Editing*
Gene Knockout Techniques
Genetic Predisposition to Disease*
HEK293 Cells
Humans
Induced Pluripotent Stem Cells / metabolism
Mutagenesis, Insertional / genetics
Neurons / metabolism*
Neurons / pathology*
Phenotype

Abstract

Publication types

MeSH terms

Grants and funding