Modification of postsynaptic genes using CRISPR/Cas9 system*

Y.K. Ko, Cathy McLaughlin, David Kerrigan, Seth G.N. Grant and Noboru H. Komiyama

Centre for Clinical Brain Sciences, The University of Edinburgh

The N-methyl-D-aspartate receptor (NMDA receptor) and its interacting proteins constitute large macro-molecular complexes (NMDAR complexes) at excitatory synapses(1). Recent human genomic studies discovered several mutations in the genes encoding the components of NMDAR complexes in various neuropsychiatric disorders including intellectual disability (ID), autism and schizophrenia. 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 adapted CRISPR 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 indicated in autism(2) and schizophrenia(3).  CRISPR-induced knockout mutation of Shank3 was successfully introduced in ES cells, and these cells were injected into blastocysts and 3 chimaeras were born. These chimaeras then were crossed with wild-type mice and germline transmission was confirmed. Having established a knockout mutation using CRISPR, we moved on to modify Synaptic GTPase-activating protein (SynGAP), another component of NMDAR complexes. De novo mutations of SynGAP have been found in ID(4), autism(4) and schizophrenia(5). To introduce a defined mutation in SynGAP gene, we designed a CRISPR-mediated point mutation, and obtained ES cells with the precise mutation. Here we demonstrate CRISPR facilitates the disruption of Shank3, as well as the precise editing in SynGAP in mouse ES cells with high efficiency.

References

  1. Frank et al. Nat. Commun. 7, 11264 (2016)
  2. Durand et al. Nat. Genet. 39, 25–27 (2007)
  3. Gauthier et al. Proc. Natl. Acad. Sci. 107, 7863–7868 (2010)
  4. Berryer et al. Hum. Mutat. 34, 385–394 (2013)
  5. Purcell et al. Nature 506, 185–90 (2014)

Funded by:  YKK is funded by Principal’s Career Development PhD Scholarship and Edinburgh Global Research Scholarship from the University of Edinburgh and the Genes to Cognition (G2C) programme

* entered into the PhD student poster competition