One-Day TALEN Assembly Protocol and a Dual-Tagging System for Genome Editing

[Image: see text] This study developed a new rapid transcription activator-like effector nuclease (TALEN) preparation protocol by thoroughly redesigning the widely used Golden Gate TALEN and TAL Effector Kit 2.0. The new protocol can be used to prepare any custom 18-bp binding TALENs in just one day (about 12 h), more rapidly than CRISPR. This protocol used a set of linear monomers, a final TALE-FokI backbone plasmid, and a pipeline to assemble the ready-to-use TALEN expression plasmid, which were all newly developed for this study. The set of linear monomers can be easily produced and reproduced by high-fidelity polymerase chain reaction (PCR) amplification in a 96-well plate using a pair of universal primers. Most important of all, our rapid TALEN construction pipeline can easily obtain many positive colonies with high efficiency (over 80%). By preparing five pairs of TALENs targeting five NF-κB genes (RELA, RELB, CREL,NFKB1, and NFKB2) and editing these genes in different cell lines (293T, HepG2, and PANC1), this study demonstrated that the new protocol has high efficiency, reproducibility, reliability, and applicability. Moreover, this study showed that the fabricated TALEN has much higher editing efficiency than CRISPR. Finally, this study developed a dual-tagging system for simultaneously tagging target proteins and successfully edited cells with a streptavidin-binding peptide (SBP) or AviTag via homology-directed repair, which could have wide applications in protein (antigen) preparation, immunoprecipitation, and a transcription factor chromatin immunoprecipitation assay.