Reprogramming homing endonuclease specificity through computational design and directed evolution.

TitleReprogramming homing endonuclease specificity through computational design and directed evolution.
Publication TypeJournal Article
Year of Publication2013
AuthorsThyme, S. B., Boissel S. J. S., Arshiya Quadri S., Nolan T., Baker D. A., Park R. U., Kusak L., Ashworth J., & Baker D.
JournalNucleic acids research
Date Published2013 Nov 21
ISSN1362-4962
KeywordsPrimary Publication
Abstract

Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally.

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http://www.ncbi.nlm.nih.gov/pubmed/24270794?dopt=Abstract

Alternate JournalNucleic Acids Res.