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Comprehensive analysis of the specificity of transcription activator-like effector nucleases

2014; Oxford University Press; Volume: 42; Issue: 8 Linguagem: Inglês

10.1093/nar/gku155

1362-4962

Alexandre Juillerat, Gwendoline Dubois, Julien Valton, Séverine Thomas, Stefano Stella, Alan Maréchal, Stéphanie Langevin, Nassima Benomari, Claudia Bertonati, George H. Silva, Fayza Daboussi, Jean-Charles Epinat, Guillermo Montoya, Aymeric Duclert, Philippe Duchâteau,

RNA and protein synthesis mechanisms

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.