Cas9/gRNA RNP VLP was generated as described previously [22 (link)]. Briefly, 293T cells were seeded in a 10 cm dish with DMEM medium supplemented with 10% fetal bovine serum. After 24 h, the culture medium was replaced with 8 ml of Opti-MEM. A total of 7.5 µg of plasmids expressing brachyury targeting gRNAs a + c, b + c, a + d, or b + d (See Fig. 1A) were mixed with 7.5 µg of COM-modified packaging plasmid pspAX2-D64V-NC-COM and 7.5 µg of plasmid pMDLg (Addgene, Watertown, MA, USA) in 600 µl Opti-MEM. 40 µl of Fugene HD was then be added and incubated at room temperature for 10 min. The mixture was added into 293T cells. After 24-h transfection, the culture medium was changed with 15 ml of Opti-MEM. The supernatant containing Cas9 RNP VLP was collected after an additional 48-h transfection and spun for 5 min at 500 g to remove cell debris. The Cas9/gRNA RNP VLP in the supernatant was concentrated by ultracentrifugation at 100,000 g for 2 h at 4 °C. VLP pellets were resuspended in PBS. Cas9/gRNA RNP VLP was quantified by p24 based ELISA according to the manufacturer’s instructions.
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