title: CRISPR-Cas9-based knockout of the prion protein and its effect on the proteome.
abstract: The molecular function of the cellular prion protein (PrPC) and the mechanism by which it may contribute to neurotoxicity in prion diseases and Alzheimer's disease are only partially understood. Mouse neuroblastoma Neuro2a cells and, more recently, C2C12 myocytes and myotubes have emerged as popular models for investigating the cellular biology of PrP. Mouse epithelial NMuMG cells might become attractive models for studying the possible involvement of PrP in a morphogenetic program underlying epithelial-to-mesenchymal transitions. Here we describe the generation of PrP knockout clones from these cell lines using CRISPR-Cas9 knockout technology. More specifically, knockout clones were generated with two separate guide RNAs targeting recognition sites on opposite strands within the first hundred nucleotides of the Prnp coding sequence. Several PrP knockout clones were isolated and genomic insertions and deletions near the CRISPR-target sites were characterized. Subsequently, deep quantitative global proteome analyses that recorded the relative abundance of>3000 proteins (data deposited to ProteomeXchange Consortium) were undertaken to begin to characterize the molecular consequences of PrP deficiency. The levels of ∼ 120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins belonging to extracellular components, cell junctions or the cytoskeleton.
authors.affiliation: Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Mohadeseh
authors.initials: M
authors.lastname: Mehrabian
authors.affiliation: Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Dylan
authors.initials: D
authors.lastname: Brethour
authors.affiliation: Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Sarah
authors.initials: S
authors.lastname: MacIsaac
authors.affiliation: Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jin Kyu
authors.initials: JK
authors.lastname: Kim
authors.affiliation: Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: C Geeth
authors.initials: CG
authors.lastname: Gunawardana
authors.affiliation: Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Hansen
authors.initials: H
authors.lastname: Wang
authors.affiliation: Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Gerold
authors.initials: G
authors.lastname: Schmitt-Ulms
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D051379:Mice; D011328:Prions / Q000235:genetics*; D020543:Proteome
chemical_list: D011328:Prions; D020543:Proteome