title: Comprehensive analysis of the specificity of transcription activator-like effector nucleases.
abstract: A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.
authors.affiliation: Cellectis S.A., 8 Rue de la Croix Jarry, 75013 Paris, France, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre, Macromolecular Crystallography Group, c/Melchor Fdez. Almagro 3, 28029 Madrid, Spain and Structural Biology Group, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Juillerat
authors.forename: Gwendoline
authors.initials: G
authors.lastname: Dubois
authors.forename: Julien
authors.initials: J
authors.lastname: Valton
authors.forename: Séverine
authors.initials: S
authors.lastname: Thomas
authors.forename: Stefano
authors.initials: S
authors.lastname: Stella
authors.forename: Alan
authors.initials: A
authors.lastname: Maréchal
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Langevin
authors.forename: Nassima
authors.initials: N
authors.lastname: Benomari
authors.forename: Claudia
authors.initials: C
authors.lastname: Bertonati
authors.forename: George H
authors.initials: GH
authors.lastname: Silva
authors.forename: Fayza
authors.initials: F
authors.lastname: Daboussi
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Epinat
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
authors.forename: Aymeric
authors.initials: A
authors.lastname: Duclert
authors.forename: Philippe
authors.initials: P
authors.lastname: Duchateau
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation; D040081:Protein Array Analysis; D015202:Protein Engineering; D015003:Yeasts / Q000235:genetics
chemical_list: D000596:Amino Acids; D004268:DNA-Binding Proteins; D004247:DNA; D003851:Deoxyribonucleases