TY - JOUR
T1 - BuD, a helix-loop-helix DNA-binding domain for genome modification
AU - Stella, Stefano
AU - Molina, Rafael
AU - López-Méndez, Blanca
AU - Juillerat, Alexandre
AU - Bertonati, Claudia
AU - Daboussi, Fayza
AU - Campos-Olivas, Ramon
AU - Duchateau, Phillippe
AU - Montoya, Guillermo
PY - 2014/1/1
Y1 - 2014/1/1
N2 - 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.
AB - 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.
KW - gene targeting
KW - genetics
KW - protein-DNA interaction
UR - http://www.scopus.com/inward/record.url?scp=84903975507&partnerID=8YFLogxK
U2 - 10.1107/s1399004714011183
DO - 10.1107/s1399004714011183
M3 - Journal article
C2 - 25004980
AN - SCOPUS:84903975507
SN - 2059-7983
VL - 70
SP - 2042
EP - 2052
JO - Acta Crystallographica Section D: Structural Biology
JF - Acta Crystallographica Section D: Structural Biology
IS - 7
ER -