Figure 12.

GFP disruption mediated by WT and RR Cpf1 variants in HEK-293.GFP cells. GFP disruption mediated by WT (black) and RR (striped) Cpf1 variants on targets with (A) TYTV, (B) TTYV or (C) TTTV/TCCV PAM sequences (TTTV PAM: GFP target 1–2, gray background; TCTV PAM: GFP target 21–24, TTCV A PAM: GFP target 25–28, TCCV PAM: GFP target 29–32, no background). The nuclease vector along with the corresponding crRNA expressing vector were transfected into HEK-293.GFP cells and GFP positive cells were counted 7 days post-transfection by flow cytometry. For controls we used either inactive Cpf1 expressing nuclease vectors or no-target crRNA expressing vectors. GFP disruption activities of the nucleases were calculated as follows: the measured percentage of GFP-positive cells for each sample was subtracted from the total average obtained with the controls. Bars correspond to averages of n = 3 parallel samples.