title: Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases.
abstract: BACKGROUND
  The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases.
  
  
  RESULTS
  In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines.
  
  
  CONCLUSIONS
  Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases.
authors.affiliation: CELLECTIS S,A, 8 Rue de la Croix Jarry, Paris 75013, France. alexandre.juillerat@cellectis.com.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Juillerat
authors.forename: Marine
authors.initials: M
authors.lastname: Beurdeley
authors.forename: Julien
authors.initials: J
authors.lastname: Valton
authors.forename: Séverine
authors.initials: S
authors.lastname: Thomas
authors.forename: Gwendoline
authors.initials: G
authors.lastname: Dubois
authors.forename: Mikhail
authors.initials: M
authors.lastname: Zaslavskiy
authors.forename: Jérome
authors.initials: J
authors.lastname: Mikolajczak
authors.forename: Fabian
authors.initials: F
authors.lastname: Bietz
authors.forename: George H
authors.initials: GH
authors.lastname: Silva
authors.forename: Aymeric
authors.initials: A
authors.lastname: Duclert
authors.forename: Fayza
authors.initials: F
authors.lastname: Daboussi
authors.forename: Philippe
authors.initials: P
authors.lastname: Duchateau
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D056426:Genetic Loci; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific