title: Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity.
abstract: Transcription activator-like effector (TALE) nuclease (TALEN) is a site-specific nuclease, which can be freely designed and easily constructed. Numerous methods of constructing TALENs harboring different TALE scaffolds and repeat variants have recently been reported. However, the functionalities of structurally different TALENs have not yet been compared. Here, we report on the functional differences among several types of TALENs targeting the same loci. Using HEK293T cell-based single-strand annealing and Cel-I nuclease assays, we found that TALENs with periodically-patterned repeat variants harboring non-repeat-variable di-residue (non-RVD) variations (Platinum TALENs) showed higher activities than TALENs without non-RVD variations. Furthermore, the efficiencies of gene disruption mediated by Platinum TALENs in frogs and rats were significantly higher than in previous reports. This study therefore demonstrated an efficient system for the construction of these highly active Platinum TALENs (Platinum Gate system), which could establish a new standard in TALEN engineering.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ochiai
authors.forename: Takehito
authors.initials: T
authors.lastname: Kaneko
authors.forename: Tomoji
authors.initials: T
authors.lastname: Mashimo
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tokumasu
authors.forename: Yuto
authors.initials: Y
authors.lastname: Sakane
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Suzuki
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Miyamoto
authors.forename: Naoaki
authors.initials: N
authors.lastname: Sakamoto
authors.forename: Shinya
authors.initials: S
authors.lastname: Matsuura
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D003851:Deoxyribonucleases / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics; D018390:Gene Targeting / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D051381:Rats; D014982:Xenopus laevis / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA; D003851:Deoxyribonucleases; D004720:Endonucleases; C406922:CEL I nuclease