Abstract

BuD, A Helix-loop-helix DNA-binding Domain for Genome Modification

Stefano Stela, Associate Professor, University Of Copenhagen

Precise DNA editing offers new possibilities in synthetic biology and biomedicine to modulate or modify cellular functions. The key players in this process, to mark where the editing event should occur, are the customizable molecular tools that are able to recognize the desired DNA target. Molecules such as TALEN and CRISPR/cas9 have been used in the recent years in many DNA editing studies. In the search of new molecules to increase the repertoire of customizable DNA-binding tools we have characterized a new domain called BurrH. This protein recognizes a 19 bp DNA target, its apo and DNA-bound crystal structures reveal a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), similar to TALE. The DNA target sequence is specified by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin (HBB) gene close to mutations responsible for sickle-cell anaemia. . Furthermore, biophysical analyses by Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) reveal different mechanism of interaction between the BurrH and the DNA target, compered to the other TALE described before. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.