Figure 2.

Overview of the Study Design

(A) Fibroblasts were collected from human APPswe carriers and their non-affected relatives. The cells were transfected with S. pyogenes Cas9-2A-GFP and gRNAs. Successfully transfected cells were identified by GFP expression and sorted by FACS. Next, these cells were expanded in culture and analyzed by sequencing, western blot (WB) (for intracellular [IC] levels of APP), and ELISA (for extracellular [EC] secretion of Aβ40 and Aβ42). (B) Embryos from time-pregnant Tg2576 APPswe transgenic mice (embryonic day 14 [E14]–17) were used to generate primary cortical neuronal culture. The transgenic cultures were co-transduced for one day with AAV1-Cas9 and either AAV1-gRNA(SW1) at 3 days in vitro (DIV). At 21 DIV, the cells were collected for sequencing. (C) Adult Tg2576 transgenic mice were co-transduced unilaterally in hippocampus with AAV9-Cas9 and AAV9-gRNA(SW1). After 1 or 2 months, the mice were sacrificed and the injected hippocampi and non-injected cerebelli (as controls) were isolated for genomic DNA sequencing.