Golden gate talen and tal effector kit 2

Mutant medaka in which an ERE sequence in the npba promoter was deleted (ΔERE) were generated by TALEN-mediated genome editing. TALENs were designed to target the ERE that has been shown to be functional in vitro (Hiraki et al., 2014 (link)). The position and sequence of the targeted ERE and TALEN binding sites are shown in Figure 1—figure supplement 1. The TALE repeat arrays were assembled by the Golden Gate method (Cermak et al., 2011 (link)) as described elsewhere (Ansai et al., 2013 (link)), using the Golden Gate TALEN and TAL Effector Kit 2.0 (Addgene 1000000024). TALEN mRNAs were synthesized by in vitro transcription using the mMessage mMachine SP6 Kit (Thermo Fisher Scientific) and microinjected into the cytoplasm of embryos at the one-cell stage. Potential founders were screened by outcrossing to wild-type fish and testing progeny for mutations at the target site using a mismatch-sensitive T7 endonuclease I assay (Kim et al., 2009 (link)) followed by direct sequencing. A founder was identified that yielded a high proportion of progeny carrying a 7 bp deletion; the progeny were subsequently intercrossed to generate fish homozygous for the deletion. The genotype of each fish was determined by direct sequencing using the primers listed in Supplementary file 2. In all analyses of ∆ERE mutants, their wild-type siblings were used as controls.

For human cell and frog experiments, a two-step Golden Gate assembly method using the Platinum Gate TALEN Kit (Addgene; cat#1000000043)15 (link) was used to construct Platinum TALEN plasmids containing the homodimer-type FokI nuclease domain. Briefly, single DNA-binding repeats were assembled into the intermediate array vectors. The assembled repeat arrays were subsequently inserted into the final destination vectors, ptCMV-153/47-VR. For the silkworm experiments, the Golden Gate TALEN and TAL Effector Kit 2.0 (Addgene; cat#1000000024)31 (link) were used to construct TALENs (BLTS-5A and BLTS-4B18 (link)), and the repeat arrays were inserted into the scaffold plasmid, pBlue-TAL18 (link).

TALENs, targeting 23 different genes (including rb1, puma, mdm4, and cyclin g1), were designed and selected using the online tool TAL Effector Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu/node/add/talen) [42] (link). The Golden Gate TALEN and TAL Effector Kit 2.0 (Catalog number 1000000024) was purchased from Addgene, and TALE repeats were assembled as instructed in the Addgene protocol based on the publication by Cermak et al (2011). Briefly, the TALENs were constructed by combining the desired TAL repeats and performing several cycles of digestion and ligation. These recombined vectors were transformed into Mach1 chemically competent cells to obtain plasmids that could then be digested and ligated into TALEN expression vectors pCS2TAL3DD and pCS2TAL3RR [14] (link)) that contained the Tal constant region, golden gate cloning region and the left or right Fok1 enzyme. Golden gate clones plasmids were used for mRNA synthesis.