MegaTev activity in vitro and in yeast. (A) (top) Schematic for the modular MegaTev fusions, consisting of the I-TevI nuclease domain and various linker lengths fused to a meganuclease. Catalytically inactive R27A I-TevI and E22Q I-OnuI E2 are denoted by xTev and xOnu, respectively. (Bottom) The modular composition of the target site consisting of the 5′-CNNNG-3′ (CAACG) cleavage motif and variable length spacer region (11–21 bp) from the native I-TevI thymidylate synthase (td) gene located upstream of the meganuclease binding site. The arrows highlight the top and bottom I-TevI cleavage sites. Highlighted in the box is the DdeI site adjacent to the I-TevI cleavage site used to identify mutagenic events in HEK 293T cells. (B) Boxplot of the activity for Tev169-xOnu (top) and Tev169-xLtr (bottom) on various DNA spacer lengths in the yeast recombination assay. TO15CS(−), AAACA cleavage motif in the TO15 target site; TL15CS(−), AAACA cleavage motif in the TL15 target site; xLtr, a catalytically inactive E29Q I-LtrI. Activity was normalized to the homodimeric FokI-Zif268 fusion and data are plotted with SD for n = 3. (C) Schematic of substrate and cleavage products containing the TO15 target site indicated by a shaded rectangle. The black and grey arrows indicate the top and bottom cleavage sites (CS) for I-TevI and I-OnuI E2, respectively. Black dashed lines indicate I-TevI cleavage products (TP1 and TP2), I-OnuI E2 cleavage products (OP1 and OP2), and the internal product (IP) from both I-TevI and I-OnuI cleavages. Sizes of the substrate and products are indicated in base pairs (bp). (D) Polyacrylamide gel of internally labeled TO15 PCR substrate (SUB) incubated with (+) or without (−) purified dual active Tev169-Onu and single active site variants. The MegaTevs were incubated with substrate for the indicated times, and cleavage products are indicated on the right side of the gel based on their predicted sizes from panel (C). The sizing standard (in bp) was cropped from the gel image.