Precise and targeted genome editing using Transcription Activator-Like Effector Endonucleases (TALENs) has been widely used and proven to be an extremely effective and specific knockout strategy in both cultured cells and animal models. The current chapter describes a protocol for the construction and generation of TALENs using serial and hierarchical digestion and ligation steps, and using the synthesized TALEN pairs to achieve locus-specific targeted gene editing in mammalian cell lines using a modified clonal selection strategy in an easy and cost-efficient manner.
Keywords: Double-strand break (DSB) repair; Fok1; Gene knockout in mammalian cells; Genome engineering; Homology-directed repair (HDR); Nonhomologous end joining (NHEJ); Repeat variable di-residues (RVDs); Restriction enzyme and ligation (REAL); T7E1 assay; TALEN.