FIGURE 4.

In vitro activity of cellular Cpf1 proteins: (A) AsCpf1-MBP protein purification: The HEK293 AsCpf1-MBP cellular extract (Ext.) was analyzed on an 8% denaturing acrylamide gel, together with the Flow through (F.T.), washing steps (1, 2, and 3), and Elution of the affinity chromatography with amylose beads. The black triangle indicates the position of the migration of the AsCpf1-MBP protein (170kDa). (B) In vitro activity of Cpf1-MBP purified proteins: Cleavage activity of Cpf1-MBP proteins expressed or not with the crRNA was performed with pGFP-SSA 5′-TTTa-3′ linearized XmaI target plasmid. The plasmid substrate was digested with the EcoRI restriction enzyme to control the size of the products. (C) Dose response activity of the purified Cpf1-MBP/crRNA complex. The quantity of proteins in the sample was assessed by SDS Page electrophoresis (upper panel) and tested for DNA cleavage activity (lower panel).