Elucidation of DNA binding modes of TALEs and creation of artificial DNA binding proteins

2014 Fiscal Year Final Research Report

• Project/Area Number: 24510295
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Living organism molecular science
• Research Institution: Kyoto University
• Principal Investigator: IMANISHI Miki 京都大学, 化学研究所, 助教 (80362391)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Keywords: DNA binding protein / Directed evolution

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.

Free Research Field

生体関連化学