Abstract
The mammalian prion protein (PrP, encoded by Prnp) is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9), have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs). It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program.
Highlights
PrP is an extracellular, membrane-anchored glycoprotein, abundantly expressed in brain of a wide range of species, especially mammals [1]
fluorescence-activated cell sorting (FACS) analysis showed higher mean fluorescent intensity (MFI) for cells transfected with functional synergistic activation mediators (SAMs) compared to cells transfected but without the single guide RNA (sgRNA), indicating that CC9 induced prion protein gene (Prnp) transactivation leads to increased levels of extracellular PrP (Fig 5C)
Pathological conformers of PrP cause a wide range of fatal and infectious neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs)
Summary
PrP is an extracellular, membrane-anchored glycoprotein, abundantly expressed in brain of a wide range of species, especially mammals [1]. Despite some significant improvements [32,37,38,39], the low efficiency of modifying Prnp in ESCs with homologous recombination (HR) has long thwarted efforts with this procedure, most likely due to a “closed” chromatin state of the locus [40,41,42]. To avoid this difficulty, most investigators continue to develop PrP transgenic mice with RITs, often driven by the moPrP.XhoI vector composed of fragments of Prnp from multiple sources [43,44,45]. Improved Prnp targeting will greatly facilitate production of more consistent disease models, and Prnp-directed SAMs can complement existing models by accelerating PrP expression ubiquitously, or in specific cell populations
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