Fig. 6. Cas9 production and localisation in transfected HEK293 cells using plasmid CRISPR-C7.

a Untreated HEK293 cells acted as a negative control and Lipofectamine 3000 was used as the commercial reagent comparison. Samples were fixed 72 h post transfection and stained with Cas9 antibody in situ (red), and with DAPI as the nuclear stain. Magnification at 40×, scale bar 20 µm. b Representative western immunoblot for intracellular levels of Cas9 at the optimal conditions from transfections with HPAE-EB compared with the commercial reagent Lipofectamine 3000. Substantial Cas9 production was achieved in HEK293 cells following transfections with HPAE-EB at w/w ratios of 20:1 and 30:1. No expression of Cas9 was present in untreated or pDNA-only treated cells. GAPDH was used as the loading control. Data are representative of three independent experiments (n = 3).