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

Research output: Contribution to journalJournal articleResearchpeer-review

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

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.

Original languageEnglish
JournalNucleic Acids Research
Volume42
Issue number8
Pages (from-to)5390-402
Number of pages13
ISSN0305-1048
DOIs
Publication statusPublished - Apr 2014

    Research areas

  • Amino Acids, Animals, Base Sequence, CHO Cells, Cricetinae, Cricetulus, DNA, DNA Cleavage, DNA-Binding Proteins, Deoxyribonucleases, Mutation, Protein Array Analysis, Protein Engineering, Yeasts

ID: 138735073