BuD, a helix-loop-helix DNA-binding domain for genome modification.

Stefano Stella,Rafael Molina,Fayza Daboussi,Alexandre Juillerat,Phillippe Duchateau,Claudia Bertonati,Blanca López-Méndez,Ramon Campos-Olivas,Guillermo Montoya

doi:10.1107/s1399004714011183

Abstract

DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein-DNA interactions in protein scaffolds is key to providing `toolkits' for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix-loop-helix modular domain (BurrH domain; BuD), which identifies the DNA target 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 β (HBB) gene close to mutations responsible for sickle-cell anaemia. 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.

Highlights

IntroductionThe tailoring of homing endonucleases (HEs; Redondo et al, 2008; Munoz et al, 2011) and other custom-made proteins, such as zinc fingers (ZFs; Urnov et al, 2010), transcription activator-like effector domains (TALEs; Miller et al, 2011) and the recently introduced CRISPR/Cas systems (Cong et al, 2013; Mali et al, 2013), has demonstrated the potential of this approach to create new specific instruments to target genes for activation, repression or repair (Prieto et al, 2012)
New residues (Thr and Arg) at the 13th position of the repeat, which could potentially be involved in DNA recognition, suggested the presence of new interactions involved in determining protein–DNA specificity
A structural comparison of BurrH protein with transcription activator-like effector domains (TALEs) suggests that protein evolution has generated a structural helix–loop–helix motif to create a modular DNA-binding domain

Summary

Introduction

The tailoring of homing endonucleases (HEs; Redondo et al, 2008; Munoz et al, 2011) and other custom-made proteins, such as zinc fingers (ZFs; Urnov et al, 2010), transcription activator-like effector domains (TALEs; Miller et al, 2011) and the recently introduced CRISPR/Cas systems (Cong et al, 2013; Mali et al, 2013), has demonstrated the potential of this approach to create new specific instruments to target genes for activation, repression or repair (Prieto et al, 2012) These tools will be important in organism design and medical applications, where they can be applied as ex vivo therapies in human monogenic diseases (Redondo et al, 2008).

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Conclusion