C57BL/6- Prnp^ZH3/ZH3 mice do not have TALEN off-target cleavage sites. T7 endonuclease I digestion of PCR products generated from predicted OTs (Table S1) and Prnp as a digestion positive control. Analyses were performed on the founder Prnp^WT/ZH3 mouse and one control C57BL/6J mouse. Before enzymatic digestion, amplicons were subjected to a temperature gradient enabling the formation of heteroduplexes in the presence of heterozygous mutations in the amplified gDNA. In the presence of TALEN-induced mutations, fragments of the size indicated below the gels are expected to appear, in addition to the undigested, WT amplicon. Nonconsecutive lanes from the same gel show Prnp amplicon as a control. Only in the founder Prnp^WT/ZH3 mouse T7 endonuclease I digestion of the Prnp amplicon results in the formation of the two predicted fragments (indicated by an asterisk).