1 Protein Structure and Function Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet2 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet3 unknown4 Protein Structure and Function Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet
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.
Nucleic Acids Research, 2014, Vol 42, Issue 8, p. 5390-402
Amino Acids; Animals; Base Sequence; CHO Cells; Cricetinae; Cricetulus; DNA; DNA Cleavage; DNA-Binding Proteins; Deoxyribonucleases; Mutation; Protein Array Analysis; Protein Engineering; Yeasts