Figure 1

Design, creation and In vivo characterization of the three nuclease architectures based on the FokI catalytic domain in yeast. (A) Schematic representation of the two different scaffolds used in this study including the positions of N and C-terminal domains, DNA binding domain as well as the Tail and Head positions. (B) Schematic representation of the yeast extrachromosomal single strand annealing (SSA) assay. The reporter plasmid containing a Nuclease Target Sequence (NTS) is flanked by overlapping truncated LacZ genes sequences. Cleavage of the target sequence in yeast leads to the restoration of the LacZ marker through the single strand annealing (SSA) pathway of recombination. The restoration of the functional LacZ gene is quantified by a β-galactosidase activity assay and related to the nuclease efficiency. (C, D and E) Representative examples of activity measurements from the yeast SSA assay for the three architectures obtained on the same filter. (C) HtH architecture where two FokI::TALE scaffolds are facing each other on the two DNA strands and in a head to head orientation. (D) Classical TtT architecture where two TALE::FokI scaffolds are facing each other on the two DNA strands and in a Tail to Tail orientation. (E) TtH architecture where a TALE::FokI and a FokI::TALE scaffolds are facing each other on the same DNA strand and in a Tail to Head orientation. The nuclease activity measured for the three architectures in yeast using the single strand annealing assay (SSA) as a function of target spacer length (5-35 bp) is displayed at the bottom of each figure panels. For each filter, three controls (negative control, weak nuclease and strong nuclease) were measured multiple times (n > 100). Standard deviation on these activity measurements were typically of 0.05.