Since ESCs divide ~ three times per day, we used a murine N2a neuroblastoma cell line since they divide far less frequently and will allow for the CC9 components to be expressed and then potentially regulate Prnp. N2A cells were transfected with combinations of vectors encoding 1) dCas9-VP64 fusion, 2) MS2–p65–HSF1 fusion protein and 3) sgRNA2.0 backbone containing MS2 stem loops and test gRNA spacers (Fig 4B) [49]. Here, HSF1 is linked to neuroblastoma.