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Title: Generation of mouse models of neurodevelopmental disorders using the CRISPR/Cas9 system
Author: Ko, Yoo Koung
ISNI:       0000 0004 7969 1421
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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The N-methyl-D-aspartate receptors (NMDARs) and its interacting proteins constitute large macro-molecular complexes (NMDAR complexes) at glutamate synapses. Recent human genomic studies discovered many mutations in the genes encoding the components of NMDAR complexes in various neurodevelopmental disorders. However, little is known about how mutations in these genes alter molecular and cellular pathways leading to pathological phenotypes. To answer the question, our lab has been systematically mutating synaptic genes in mice using conventional gene targeting methods. To further accelerate generation of mutants we adopted the CRISPR/Cas9 genome editing system in mouse embryonic stem (ES) cells. SH3 and multiple ankyrin repeat domains 3 (SHANK3) is a crucial scaffolding protein of NMDAR complexes and is implicated in autism spectrum disorders, intellectual disability, and schizophrenia. A CRISPR/Cas9-induced knockout mutation of SHANK3 was successfully introduced in ES cells, and these cells were injected into mouse blastocysts and three chimaeras were born. These chimaeras were then crossed with wild-type mice and germline transmission was confirmed. Having established a knockout mutation using the CRISPR/Cas9 system, we moved on to modify Synaptic GTPase-activating protein 1 (SYNGAP1), another component of NMDAR complexes. De novo mutations of SYNGAP1 have been found in intellectual disability, autism spectrum disorders, and schizophrenia. To introduce a defined mutation in the SYNGAP1 gene, we designed a CRISPR/Cas9-mediated point mutation, and ES cell lines with the precise mutation were obtained. These cell lines were further developed to create a novel animal colony. Here we demonstrate CRISPR/Cas9 facilitates disruption of SHANK3, as well as the precise editing in SYNGAP1 in mouse ES cells with high efficiency. Furthermore, we report successful generation and characterisation of genome-engineered mice using CRISPR/Cas9-modified ES cells.
Supervisor: Komiyama, Noboru ; Grant, Seth Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: synaptic gene mutations ; mouse model ; CRISPR/Cas9 ; autism-associated gene ; SHANK3 ; SYNGAP1 ; neurodevelopmental disorders