AbstractsBiology & Animal Science

Generation andcharacterization of transcription activator-like effectornucleases

by Laura Waltl




Institution: Universität Giessen
Department:
Year: 2016
Posted: 02/05/2017
Record ID: 2131338
Full text PDF: http://geb.uni-giessen.de/geb/volltexte/2016/12099


Abstract

Designer nucleases are developed to modify the genome in various organisms, and are promising molecular tools for the advancement of gene therapy. They should introduce only one specific double-strand or single-strand break in a complex genome in order to trigger the DNA repair mechanisms of the cell such that the DNA damage is repaired by homologous directed repair (HDR) or non-homologous end joining (NHEJ), an error-prone repair mechanism that can be applied to knock out genes. With the addition of an external DNA fragment, HDR can be exploited to correct or add a gene. Transcription activator-like effector (TALE) nucleases belong to a platform of designer nucleases and are known to be highly specific, efficient and easy to customize for any given target. Nevertheless, there is still a need to improve the accuracy of TALE nucleases for their reliable application in vivo. To this end, off-target activity should be avoided without compromising cleavage efficiency. This can be achieved by optimization of the catalytic domain as well as by improving the binding domain. The subjects of this comparative study are the homing endonuclease I-SceI, the widely used TALE-FokI fusion protein, the new RNA guided Cas9 nuclease and our recently developed TALE-PvuII variants as well as the TALE-MutH fusion protein, whose nuclease domain is a sequence- and strand-specific nickase. In the study we have analyzed the toxicity and cleavage efficiency of these specific nucleases in a yeast based single-strand annealing assay as well as in vitro. The present study also focus on the characterization of the in vitro binding mechanism of TALE. Binding characteristics of AvrBs3(Delta N152-C28) were analyzed via anisotropy measurements, specific singleand double-labeled TALE variants were designed and DNA::Protein complexes were analyzed employing FRET measurements.We gained labeled variants remaining their binding characteristics and enables the investigation of the in vitro binding mechanism of TALE proteins.Our results demonstrate convincingly that the monomeric TALEMutH nickase displays less toxicity in Saccharomyces cerevisiae compared to the widely used dimeric TALE-FokI nuclease and is able to introduce the repair of the DNA damage via single-strand annealing as efficient as I-SceI, the gold standard for cleaving genomic DNA. To improve the design of TALE-MutH nickases, we analyzed the cleavage behavior on various specific loci in vitro and in vivo. The results presented show that the TALE-MutH fusion protein is a promising programmable nickase for in vivo applications and these results were confirmed by a TALE-MutH addressing a relevant therapeutic gene. Konstruierte Nukleasen wurden entwickelt um das Genom in verschiedenen Organismen zu modifizieren und sind viel versprechende Werkzeuge zur Weiterentwicklung von effizienten Gentherapien. Spezifische Nukleasen sollten einen spezifischen Doppel- oder Einzelstrangbruch in dem komplexem Genom induzieren und hierdurch den DNA Reparaturmechanismus der Zelle auslösen. Die DNA wird hierbei…