Efficient TALEN Construction for Genomic Editing

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.