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Reprogramming homing endonuclease specificity through computational design and directed evolution.

Summer B Thyme1, Sandrine J S Boissel, S Arshiya Quadri

  • 1Department of Biochemistry, University of Washington, UW Box 357350, 1705 NE Pacific St., Seattle, WA 98195, USA, Graduate Program in Biomolecular Structure and Design, University of Washington, UW Box 357350, 1705 NE Pacific St., Seattle, WA 98195, USA, Graduate Program in Molecular and Cellular Biology, University of Washington, UW Box 357275, 1959 NE Pacific St., Seattle, WA 98195, USA, Department of Life Sciences, Sir Alexander Fleming Building, Imperial College London, Imperial College Road, London SW7 2AZ, UK, Department of Genetics, University of Cambridge, Downing Street, Cambridge CB1 3QA, UK, Institute for Systems Biology, 401 Terry Avenue N, Seattle, WA 98109, USA and Howard Hughes Medical Institute, University of Washington, UW Box 357350, 1705 NE Pacific St., Seattle, WA 98195, USA.

Nucleic Acids Research
|November 26, 2013
PubMed
Summary
This summary is machine-generated.

Scientists engineered novel homing endonucleases (HEs) for precise genome editing. These engineered HEs show potential for controlling malaria vectors and correcting genetic disorders like pyruvate kinase deficiency.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Bioengineering

Background:

  • Homing endonucleases (HEs) are powerful tools for targeted genome modification.
  • Applications include controlling pathogen vectors like Anopheles gambiae and correcting genetic diseases.

Purpose of the Study:

  • To create an extensive set of HE variants with novel DNA cleavage specificities.
  • To engineer HEs for specific applications in vector control and genetic disease correction.

Main Methods:

  • Integrated experimental and computational approach for HE variant creation.
  • Utilized computational modeling and an improved selection strategy optimizing specificity and activity.
  • Engineered endonucleases targeting genes related to Anopheles sterility and pyruvate kinase deficiency.

Main Results:

  • Successfully created HE variants with novel DNA cleavage specificities.
  • Engineered an endonuclease to target a gene associated with Anopheles sterility.
  • Engineered an endonuclease to cleave near a mutation causing pyruvate kinase deficiency.
  • Observed unanticipated base context-dependence affecting specificity.

Conclusions:

  • The engineered HEs demonstrate potential for precise genome editing in diverse applications.
  • Understanding base context-dependence is crucial for broader HE specificity reprogramming.
  • This work advances the engineering of homing endonucleases for gene therapy and vector control.