Site-specific nucleases (SSNs) are molecular tools to introduce DNA double-strand breaks (DSBs) at definite genomic loci. DSBs can be exploited to knock-out, delete, repair or insert genes of interest. Construction of ssns was simplified tremendously with the discovery of transcription activator-like effector (TALE) proteins. The novel TALE nucleases (TALENs) consist of a TALE-derived DNA binding domain, guiding the construct to its target site, and a nuclease domain, which is cleaving the DNA. The standard nuclease domain is the catalytic domain of type IIS restriction endonuclease FokI, which was adopted from the older zinc-finger-nuclease architecture. FokI requires dimerization for the creation of a DSB, making two TALENs necessary. Aim of this work is the replacement of this catalytic domain to generate monomeric TALENs, that simplify production and transfection. To cover nuclease domains with varying degrees of specificity, three groups were chosen: promiscuous H-N-H and DRGH nucleases (I), the more specific I-TevI catalytic domain (II) and variants of the highly-specific homing endonuclease I-CreI (III). The selected domains were adapted to the fusion scaffold via rational design strategies and tested in vitro and in vivo in several model organisms. Special properties of these domains made the generation of novel, monomeric TALENs possible. Colicin E7, Nuclease A and Endonuclease A (I) were used to create switchable TALENs and I-CreI (III) fusion yielded a highly specific construct. Investigation of the influence of the fusion terminus allowed the construction of scaffolds with multiple nuclease domains via I-TevI (II) and FokI.
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