| Budget Amount *help |
¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2012: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
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| Outline of Final Research Achievements |
Transcription activator-like effectors (TALEs) are useful templates for genome editing at specific sites. However, most TALE binding sites are preceded by a highly conserved 5’-terminal T nucleotide (5’-T), resulting in restriction of target sites. To remove this constraint, tryptophan 232 in the repeat-1 loop region of the dHax3 N-terminal domain was substituted for other 19 amino acids. Furthermore, whole hairpin loop region of repeat-1 was randomized. Although point mutation was insufficient to remove the constraint, directed evolution from the randomized library yielded repeat-1 mutants with unbiased targeting sites for 5’-terminal bases. It was indicated that the repeat-1 loop region of dHax3 is important for 5’-terminal base accommodation, and that molecular evolution of repeat-1 of TALEs is an efficient strategy to remove the constraint and thus allow targeting of any DNA sequences.
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