title: Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy.
abstract: Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats.
authors.affiliation: Research Center for the Mathematics on Chromatin Live Dynamics (RcMcD), Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Tochio
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Umehara
authors.affiliation: Research Center for the Mathematics on Chromatin Live Dynamics (RcMcD), Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Jun-Ichi
authors.initials: JI
authors.lastname: Uewaki
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Holger
authors.initials: H
authors.lastname: Flechsig
authors.affiliation: Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Kondo
authors.affiliation: Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.
authors.forename: Takehisa
authors.initials: T
authors.lastname: Dewa
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Faculty of Pharmaceutical Sciences and Center for Research and Education on Drug Discovery, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Saitoh
authors.affiliation: Research Center for the Mathematics on Chromatin Live Dynamics (RcMcD), Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Togashi
authors.affiliation: Research Center for the Mathematics on Chromatin Live Dynamics (RcMcD), Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.|Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Tate
mesh_terms: D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D000067493:Dynamic Light Scattering; D000072669:Gene Editing; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D009154:Mutation; D020449:Repetitive Sequences, Amino Acid; D013816:Thermodynamics; D000069896:Transcription Activator-Like Effector Nucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004247:DNA; D000069896:Transcription Activator-Like Effector Nucleases