Fig. 9

The self-cleaving Cas9 system. A The plasmid based self-limiting system incorporates a self-targeting guide into the Cas9 expressing plasmids. Upon expression, Cas9 assembly will generate two Cas9 complexes; one targeting and cleaving the AAVS1 genomic locus and the other cleaving the Cas9 expressing plasmid. B, C HEK293T cells were transfected with Cas9 expressing plasmids with their appropriate self-targeting guides. Cells were collected at each time points (8, 16, 24, 48, and 72 h after transfection) and nuclear extracts were obtained. Western blotting was used to detect Cas9 protein (161 kDa) or the TBP loading control (37.6 kDa) in B control and C cells transfected with Cas9 self-cleaving guides