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

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 targe...

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Bibliographic Details
Published in:Nucleic acids research 2014-04, Vol.42 (8), p.5390-5402
Main Authors: Juillerat, Alexandre, Dubois, Gwendoline, Valton, Julien, Thomas, Séverine, Stella, Stefano, Maréchal, Alan, Langevin, Stéphanie, Benomari, Nassima, Bertonati, Claudia, Silva, George H., Daboussi, Fayza, Epinat, Jean-Charles, Montoya, Guillermo, Duclert, Aymeric, Duchateau, Philippe
Format: Article
Language:English
Subjects:
Amino Acids - chemistry
Animals
Base Sequence
CHO Cells
Cricetinae
Cricetulus
Deoxyribonucleases - chemistry
Deoxyribonucleases - metabolism
DNA - chemistry
DNA - metabolism
DNA Cleavage
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - metabolism
Life Sciences
Mutation
Protein Array Analysis
Protein Engineering
Synthetic Biology and Chemistry
Yeasts - genetics
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gku155