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

Overview

Journal J Mol Biol

Publisher Elsevier

Specialties Microbiology
Molecular Biology

Date 2010 May 8

PMID 20447404

Citations 106

Authors

Jing Guo

Thomas Gaj

Carlos F Barbas 3rd

Affiliations

Soon will be listed here.

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.

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References

Urnov F, Miller J, Lee Y, Beausejour C, Rock J, Augustus S . Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005; 435(7042):646-51. DOI: 10.1038/nature03556. View

Valerie K, Povirk L . Regulation and mechanisms of mammalian double-strand break repair. Oncogene. 2003; 22(37):5792-812. DOI: 10.1038/sj.onc.1206679. View

Liu Q, Segal D, Ghiara J, Barbas 3rd C . Design of polydactyl zinc-finger proteins for unique addressing within complex genomes. Proc Natl Acad Sci U S A. 1997; 94(11):5525-30. PMC: 20811. DOI: 10.1073/pnas.94.11.5525. View

Dreier B, Beerli R, Segal D, Flippin J, Barbas 3rd C . Development of zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem. 2001; 276(31):29466-78. DOI: 10.1074/jbc.M102604200. View

Beerli R, Barbas 3rd C . Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol. 2002; 20(2):135-41. DOI: 10.1038/nbt0202-135. View

Segal D, Dreier B, Beerli R, Barbas 3rd C . Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. Proc Natl Acad Sci U S A. 1999; 96(6):2758-63. PMC: 15842. DOI: 10.1073/pnas.96.6.2758. View

Zeevi V, Tovkach A, Tzfira T . Increasing cloning possibilities using artificial zinc finger nucleases. Proc Natl Acad Sci U S A. 2008; 105(35):12785-90. PMC: 2529078. DOI: 10.1073/pnas.0803618105. View

Mandell J, Barbas 3rd C . Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 2006; 34(Web Server issue):W516-23. PMC: 1538883. DOI: 10.1093/nar/gkl209. View

Handel E, Alwin S, Cathomen T . Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity. Mol Ther. 2008; 17(1):104-11. PMC: 2834978. DOI: 10.1038/mt.2008.233. View

10.

Bae K, Kwon Y, Shin H, Hwang M, Ryu E, Park K . Human zinc fingers as building blocks in the construction of artificial transcription factors. Nat Biotechnol. 2003; 21(3):275-80. DOI: 10.1038/nbt796. View

11.

Abedi M, Caponigro G, Kamb A . Green fluorescent protein as a scaffold for intracellular presentation of peptides. Nucleic Acids Res. 1998; 26(2):623-30. PMC: 147301. DOI: 10.1093/nar/26.2.623. View

12.

Geurts A, Cost G, Freyvert Y, Zeitler B, Miller J, Choi V . Knockout rats via embryo microinjection of zinc-finger nucleases. Science. 2009; 325(5939):433. PMC: 2831805. DOI: 10.1126/science.1172447. View

13.

Gonzalez B, Schwimmer L, Fuller R, Ye Y, Asawapornmongkol L, Barbas 3rd C . Modular system for the construction of zinc-finger libraries and proteins. Nat Protoc. 2010; 5(4):791-810. PMC: 2855653. DOI: 10.1038/nprot.2010.34. View

14.

Miller J, Holmes M, Wang J, Guschin D, Lee Y, Rupniewski I . An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol. 2007; 25(7):778-85. DOI: 10.1038/nbt1319. View

15.

Perez E, Wang J, Miller J, Jouvenot Y, Kim K, Liu O . Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol. 2008; 26(7):808-16. PMC: 3422503. DOI: 10.1038/nbt1410. View

16.

Stemmer W . Rapid evolution of a protein in vitro by DNA shuffling. Nature. 1994; 370(6488):389-91. DOI: 10.1038/370389a0. View

17.

Doyon Y, McCammon J, Miller J, Faraji F, Ngo C, Katibah G . Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol. 2008; 26(6):702-8. PMC: 2674762. DOI: 10.1038/nbt1409. View

18.

Dreier B, Segal D, Barbas 3rd C . Insights into the molecular recognition of the 5'-GNN-3' family of DNA sequences by zinc finger domains. J Mol Biol. 2000; 303(4):489-502. DOI: 10.1006/jmbi.2000.4133. View

19.

Bahassi E, SALMON M, Van Melderen L, Bernard P, Couturier M . F plasmid CcdB killer protein: ccdB gene mutants coding for non-cytotoxic proteins which retain their regulatory functions. Mol Microbiol. 1995; 15(6):1031-7. DOI: 10.1111/j.1365-2958.1995.tb02278.x. View

20.

Kim Y, Cha J, Chandrasegaran S . Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A. 1996; 93(3):1156-60. PMC: 40048. DOI: 10.1073/pnas.93.3.1156. View