TALENs induce specific DNA DSBs within the MLL gene. (A) Schematic illustration of the human MLL gene shows recognition sites (bold sequences) for TALEN pairs designed to cleave within the 5′ portion of MLL intron 11. Black boxes represent respective exons of the MLL gene. (B) Gel image shows results of the T7 endonuclease assay performed on gDNA isolated from K562 or CD34⁺ cells nucleofected with the 3 different MLL TALEN pairs (P1, P2, P3) or control (GFP) as indicated. Digested PCR products (*) of the MLL locus represent the presence of strand mismatches resulting from indels that are generated during nonhomologous end joining (NHEJ) repair of DSBs. (C) DNA sequences of the amplified endogenous MLL locus resulting from the best TALEN pair (P3) show unique insertions or deletions resulting from NHEJ (K562 24 of 70: 34.3%; CD34⁺ 11 of 70: 15.7% mutation ratio). Italic letters represent insertions; underlined letters denote TALEN binding sites; red and blue text indicates MLL exon 11 and MLL intron 11, respectively. (D) Indel frequencies were measured by TIDE and compared with indel frequencies of the control sample.²¹  eff., efficiency.