Transcription activator-like effectors (TALEs) are convenient tools for genome engineering at specific genomic sites. However, their use is constrained because most TALE binding sites are preceded by a highly conserved 5' terminal T nucleotide (5'-T). To remove the 5'-T constraint, we substituted tryptophan 232 in the repeat-1 loop region of the dHax3 N-terminal domain for other amino acids. Furthermore, we randomized four amino acid residues of the hairpin loop region of repeat-1. Although point mutation was insufficient to remove the 5'-T constraint, directed evolution from the randomized library yielded repeat-1 mutants with unbiased targeting sites for 5'-bases. Our result indicates that the repeat-1 loop region of dHax3 is important for 5'-base accommodation, and that molecular evolution of repeat-1 of TALEs is an efficient strategy to remove the 5'-T constraint and thus allow targeting of any DNA sequences. (C) 2013 Elsevier Inc. All rights reserved.