Schematic diagrams of TALEN structure and PCR-based assay for detection of mutagenesis. (A) Schematic drawing of TALEN structure. TALEN architecture was optimized for both zebrafish and Xenopus. Each TALEN monomer consists of a nuclear location signal (NLS), 152 amino acids deletion N-terminal, 63 amino acids from C-terminal, the TALE repeat domains, and modified Fok I nuclease domain ELD/KKR. Each TALE repeat unit consists of 34 amino acids, in which the amino acids at positions 12 and 13 are called ‘repeat-variable di-residues’ (RVDs). The RVD determine binding specificity to DNA bases following the code that NG, NI, HD, and NN respectively recognized thymine, adenine, cytosine and guanine. (B) Schematic diagram illustrating binding of TALENs to their targeted DNA sites. Each monomer of a TALEN pair recognizes and binds DNA via upstream or downstream EBE individually. Two Fok I nuclease domains ELD and KKR dimerize and function as endonuclease, generating DNA double strands break (DSB) at spacer between the two EBE sites. (C) PCR assay determining indel mutations induced by TALENs. The DNA fragment coving two EBE sites were amplified with Primer 1/3, the amplicons were then subcloned into pMD18-T using TA cloning. Primer 1/3 were employed to check insertion of targeted sequence after TA cloning. Primer 2 covered the joint region between upstream EBE and the spacer. When indel mutations were amplified in the spacer region, no amplicons would be generated by Primer 2/3. The corresponding plasmids were then sequenced to verify TALEN-induced mutations. Primer 1/4 may also be introduced to this PCR assay for detection of some mutations close to the downstream EBE site, which could not be captured by Primer 2/3. Not drawn to scale.