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.
Highlights
The prion protein (PrP), with more than 10,000 articles published on it, several dozen ortholog PrP structures deposited in protein data bank repositories, and countless reagents in circulation to facilitate its detection and characterization, PLOS ONE | DOI:10.1371/journal.pone.0114594 December 9, 2014Changes to the Proteome by clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-Based PrP Knockout may rank amongst the most studied proteins to date
Our analysis revealed reproducibly altered the abundance levels of,120 proteins in cells that exhibit no or reduced levels of PrP
Any CRISPR-Cas9-based gene knockout experiment requires that consideration be given to the choice of cell type, the reagents and strategy employed, the method for identifying positive clones and the possibility of off-target effects
Summary
Changes to the Proteome by CRISPR-Cas9-Based PrP Knockout may rank amongst the most studied proteins to date. PrP gained its notoriety by the central role it was shown to play in a group of invariably fatal prion diseases that can afflict humans and some mammalian species [1]. The cellular form of the prion protein (PrPC), which is widely expressed in vertebrate cells, is known to undergo a conformational change and to acquire different physicochemical properties [2]. PrPC has been proposed to play a role in Alzheimer’s disease by serving as a receptor of oligomeric forms of the amyloid beta (Ab) peptide [4], the primary constituent of amyloid plaques observed in individuals afflicted with this disease. The degree to which PrPC contributes in the aforementioned neurodegenerative diseases to complex cellular etiologies that lead to neurotoxicity and, eventually, cell death has not been resolved [5]
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