Directed Evolution of an Enhanced and Highly Efficient FokI Cleavage Domain for Zinc Finger Nucleases

Directed Evolution of an Enhanced and Highly Efficient FokI Cleavage Domain for Zinc Finger... Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The high specificity and affinity of these chimeric enzymes are based on custom-designed zinc finger proteins (ZFPs). To improve the performance of existing ZFN technology, we developed an in vivo evolution-based approach to improve the efficacy of the FokI cleavage domain (FCD). After multiple rounds of cycling mutagenesis and DNA shuffling, a more efficient nuclease variant ( Sharkey ) was generated. In vivo analyses indicated that Sharkey is > 15-fold more active than wild-type FCD on a diverse panel of cleavage sites. Further, a mammalian cell-based assay showed a three to sixfold improvement in targeted mutagenesis for ZFNs containing derivatives of the Sharkey cleavage domain. We also identified mutations that impart sequence specificity to the FCD that might be utilized in future studies to further refine ZFNs through cooperative specificity. In addition, Sharkey was observed to enhance the cleavage profiles of previously published and newly selected heterodimer ZFN architectures. This enhanced and highly efficient cleavage domain will aid in a variety of ZFN applications in medicine and biology. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Biology Elsevier

Directed Evolution of an Enhanced and Highly Efficient FokI Cleavage Domain for Zinc Finger Nucleases

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Publisher
Elsevier
Copyright
Copyright © 2010 Elsevier Ltd
ISSN
0022-2836
DOI
10.1016/j.jmb.2010.04.060
Publisher site
See Article on Publisher Site

Abstract

Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The high specificity and affinity of these chimeric enzymes are based on custom-designed zinc finger proteins (ZFPs). To improve the performance of existing ZFN technology, we developed an in vivo evolution-based approach to improve the efficacy of the FokI cleavage domain (FCD). After multiple rounds of cycling mutagenesis and DNA shuffling, a more efficient nuclease variant ( Sharkey ) was generated. In vivo analyses indicated that Sharkey is > 15-fold more active than wild-type FCD on a diverse panel of cleavage sites. Further, a mammalian cell-based assay showed a three to sixfold improvement in targeted mutagenesis for ZFNs containing derivatives of the Sharkey cleavage domain. We also identified mutations that impart sequence specificity to the FCD that might be utilized in future studies to further refine ZFNs through cooperative specificity. In addition, Sharkey was observed to enhance the cleavage profiles of previously published and newly selected heterodimer ZFN architectures. This enhanced and highly efficient cleavage domain will aid in a variety of ZFN applications in medicine and biology.

Journal

Journal of Molecular BiologyElsevier

Published: Jul 2, 2010

References

  • Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases
    Bibikova, M.; Golic, M.; Golic, K.G.; Carroll, D.
  • Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases
    Doyon, Y.; McCammon, J.M.; Miller, J.C.; Faraji, F.; Ngo, C.; Katibah, G.E.
  • Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases
    Meng, X.; Noyes, M.B.; Zhu, L.J.; Lawson, N.D.; Wolfe, S.A.
  • Establishment of HIV-1 resistance in CD4 + T cells by genome editing using zinc-finger nucleases
    Perez, E.E.; Wang, J.; Miller, J.C.; Jouvenot, Y.; Kim, K.A.; Liu, O.
  • High-frequency homologous recombination in plants mediated by zinc-finger nucleases
    Wright, D.A.; Townsend, J.A.; Winfrey, R.J.; Irwin, P.A.; Rajagopal, J.; Lonosky, P.M.
  • Efficient gene targeting in Drosophila with zinc-finger nucleases
    Beumer, K.; Bhattacharyya, G.; Bibikova, M.; Trautman, J.K.; Carroll, D.
  • F plasmid CcdB killer protein: ccdB gene mutants coding for non-cytotoxic proteins which retain their regulatory functions
    Bahassi, E.M.; Salmon, M.A.; Van Melderen, L.; Bernard, P.; Couturier, M.

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