Figure 1.

Production and Characterization of Cas9 Gesicles

(A) HEK293FT producer cells were transfected with the gesicle packaging mix containing CherryPicker Red, VSV-G, Cas9, and a chosen gRNA. Gesicles were released over the course of 48 hr and were collected by media filtration and ultracentrifugation. (B) Live-cell images of HEK293FT cells either untreated (−) or transfected with the gesicle packaging mix (+). The gesicle packaging mix condition showed strong CherryPicker Red fluorescence in comparison with the untreated condition. Cell lysates were prepared from HEK293FT producer cells from untreated (−) and gesicle packaging mix (+) conditions, and a western blot was run to identify protein expression of CherryPicker Red (C), VSV-G (D), and Cas9 (E). Additionally, media were taken and concentrated to obtain gesicles from untransfected (−) or gesicle packaging mix (+) cells (Conc. Media column). We observed an increased expression of CherryPicker Red (B), VSV-G (C), and Cas9 (D) in the preparation from cells transfected with the gesicle packaging mix. A parallel preparation of gesicles was prepared using the gesicle packaging mix without the presence of A/C heterodimerizer. We observed CherryPicker Red protein expression in all preparation conditions using the gesicle packaging mix (F). The absence of A/C heterodimerizer significantly decreases the expression of Cas9 as observed by western blot (G) and densitometry (H). Data are the mean + SEM of three experiments, analyzed using a one-way ANOVA with post hoc test; *p < 0.05 versus no A/C treatment group.