Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10756830 | Biochemical and Biophysical Research Communications | 2013 | 4 Pages |
Abstract
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 5â² terminal T nucleotide (5â²-T). To remove the 5â²-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 5â²-T constraint, directed evolution from the randomized library yielded repeat-1 mutants with unbiased targeting sites for 5â²-bases. Our result indicates that the repeat-1 loop region of dHax3 is important for 5â²-base accommodation, and that molecular evolution of repeat-1 of TALEs is an efficient strategy to remove the 5â²-T constraint and thus allow targeting of any DNA sequences.
Keywords
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Shogo Tsuji, Shiroh Futaki, Miki Imanishi,