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Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation
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BuD, a helix-loop-helix DNA-binding domain for genome modification.

Stefano Stella1, Rafael Molina1, Blanca López-Méndez2

  • 1Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid, Spain.

Acta Crystallographica. Section D, Biological Crystallography
|July 10, 2014
PubMed
Summary

Researchers engineered a novel DNA-binding protein, BurrH, for precise genome editing. This breakthrough offers a new toolkit for gene targeting and modification, advancing synthetic biology and biomedicine.

Keywords:
gene targetinggeneticsprotein–DNA interaction

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Area of Science:

  • Synthetic biology
  • Genetics
  • Structural biology

Background:

  • DNA editing holds promise for synthetic biology and biomedicine but faces challenges with accuracy and potential genome damage.
  • Precise gene modification requires engineering specific protein-DNA interactions for targeted genome regulation.

Purpose of the Study:

  • To identify and characterize novel DNA-binding proteins for precise genome editing applications.
  • To engineer new DNA-binding specificities for targeted gene modification.

Main Methods:

  • Identification and structural analysis (apo and DNA-bound) of the BurrH protein.
  • Engineering of BurrH domains (BuD) into nucleases (BuDNs).
  • Testing BuDNs for gene targeting efficiency in the human hemoglobin beta (HBB) gene locus.

Main Results:

  • The BurrH protein recognizes a 19 bp DNA target via a modular helix-loop-helix domain (BuD).
  • A single residue-to-nucleotide code within BuD allows for redesign of DNA-binding specificity.
  • Engineered BuDNs demonstrated high gene targeting efficiency near HBB mutations associated with sickle-cell anemia.

Conclusions:

  • The BurrH domain (BuD) provides a versatile platform for engineering specific DNA-binding proteins.
  • BuD-derived nucleases (BuDNs) offer a highly efficient and specific tool for genome modification.
  • This technology advances gene editing capabilities for therapeutic and research applications.