Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs. (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genotyping. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type sqt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).