FIGURE 6.

Analysis of LbCpf1 RAR and RSR mutants with the 5′-TNNa-3′ SSA library. (A) Graphic representation of the Wt and mutant protein activity: the graph is summarizing the activity observed with the 16 5′-TNNa-3′ reporter vectors for LbCpf1 and each PAM recognition mutant (RSR and RAR). The transfection was performed with 100 ng of LbCpf1 expression vectors. (B) Nuclease activity: In vitro cleavage activity of Wt LbCpf1-MBP and each mutants RSR-MBP and RAR-MBP with the XmaI linearized plasmid pGFP-SSA substrates 5′-TTTa-3′, 5′-TATa-3′ and 5′-TACa-3′. One of the plasmid substrates was digested with the EcoRI restriction enzyme to control the size of the products. (C) 6TG resistant cell selection: Pictures of Hela cells, after selection with the 6TG, following the co-transfection of the LbCpf1 (Wt or the PAM recognition mutants RSR and RAR) expression vectors, and the crRNAs that target the hprt exon3 sequences flanked, respectively by the 5′-TTTa-3′, 5′-TATa-3′ and 5′-TACa-3′ PAM sequences. The left Panel is a control experiment using a guide crRNA against dnmt1 gene.