How does ApoE selectively increase APP, but not APLP1 and APLP2 transcription? To examine this question, we used CRISPR-interference (CRISPRi) mapping with guide RNAs that were targeted to APP gene promoter sequences (Fig. 5A; Larson et al., 2013). We found that of six guide RNAs tested, only one guide RNA (sg2) blocked ApoE3-induced increases in APP protein and mRNA levels (Fig. 5B, 5C, S6B). Importantly, CRISPRi with sg2 did not impair ApoE3-induced ERK1/2 phosphorylation, and had no effect on baseline APP mRNA and protein levels. Moreover, CRISPRi with sg2 abolished ApoE-induced stimulation of Aβ42 secretion from human neurons co-cultured with MEFs without altering baseline Aβ42 secretion (Fig. 5D), and decreased Aβ42 secretion from human neurons co-cultured with glia (Fig. S6C).
