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Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays

Nature Protocols volume 4, pages 1471–1501 (2009)Cite this article

Abstract

Engineered zinc-finger nucleases (ZFNs) form the basis of a broadly applicable method for targeted, efficient modification of eukaryotic genomes. In recent work, we described OPEN (oligomerized pool engineering), an 'open-source,' combinatorial selection-based method for engineering zinc-finger arrays that function well as ZFNs. We have also shown in direct comparisons that the OPEN method has a higher success rate than previously described 'modular-assembly' methods for engineering ZFNs. OPEN selections are carried out in Escherichia coli using a bacterial two-hybrid system and do not require specialized equipment. Here we provide a detailed protocol for carrying out OPEN to engineer zinc-finger arrays that have a high probability of functioning as ZFNs. Using OPEN, researchers can generate multiple, customized ZFNs in ∼8 weeks.

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Figure 1: Zinc-finger nucleases.
Figure 2: Oligomerized pool engineering (OPEN)-selection method for engineering multi-finger arrays.
Figure 3: Construction of F′-based reporter episomes required for B2H selection strains.
Figure 4: Strategy for designing binding-site oligonucleotides.
Figure 5: Schematic overview of zinc-finger library construction.
Figure 6: Schematic depicting triplicate serial dilution and triplicate spotting of dilutions on agar plates.
Figure 7: Schematic illustrating a method for pouring gradient selection plates.
Figure 8: Appearance of a typical gradient selection plate with a successful OPEN selection.

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Acknowledgements

We thank M. Eichtinger, T. Jiang and S. Pruett-Miller for technical assistance and M. Porteus for helpful discussions during early stages of protocol development. M.L.M., S.T.-B. and J.K.J. are supported by grants from the National Institutes of Health (R01 GM069906, R24 GM078369, R21 RR024189) and the MGH Pathology Service. D.F.V. is supported by the National Science Foundation (DBI 0501678). J.D.S. is supported by a Pioneer-Hi-Bred International 2008 Graduate Fellowship, the National Institutes of Health (R33 GM066387) and the Iowa State University CIAG. Note added in proof Shukla et al.78 recently demonstrated ZFN-enhanced gene targeting of an endogenous plant gene.

Author information

Authors and Affiliations

  1. Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Morgan L Maeder, Stacey Thibodeau-Beganny & J Keith Joung

  2. Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA

    Morgan L Maeder & J Keith Joung

  3. Department of Genetics, Development & Cell Biology, Roy J. Carver Co-Laboratory, Iowa State University, Ames, Iowa, USA

    Jeffry D Sander & Daniel F Voytas

  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    J Keith Joung

  5. Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA

    Daniel F Voytas

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  1. Morgan L Maeder
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  3. Jeffry D Sander
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  4. Daniel F Voytas
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Contributions

M.L.M., S.T.-B., and J.K.J. developed the OPEN selection procedure. J.D.S. and D.F.V. developed the ZiFiT software. M.L.M., S.T.-B., J.D.S., D.F.V., and J.K.J. wrote the paper.

Corresponding author

Correspondence to J Keith Joung.

Supplementary information

Supplementary Fig. 1

Full sequence of reporter plasmid pKJ1712 (8723 bp) (PDF 28 kb)

Supplementary Fig. 2

Full sequence of the B2H vector pBR-UV5-GP-FD2 (3508 bp) (PDF 24 kb)

Supplementary Fig. 3

Full sequence of the pAC-alphaGal4 vector (3428 bp) (PDF 22 kb)

Supplementary Fig. 4

Gel electrophoresis of restriction digested plasmid pKJ1712 with and without cloned target binding site oligonucleotides EcoRI/HindIII digestions of plasmid pKJ1712 with (lane 1) and without (lane 2) target binding site oligonucleotides inserted were run on a 5% polyacrylamide gel and visualized with UV light following ethidium bromide staining. (PDF 27 kb)

Supplementary Fig. 5

Sequence of EcoRI-SalI segment of the binding site reporter (PDF 15 kb)

Supplementary Fig. 6

Sequence of XbaI-BamHI fragment encoding a generic threefinger array (PDF 15 kb)

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Maeder, M., Thibodeau-Beganny, S., Sander, J. et al. Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays. Nat Protoc 4, 1471–1501 (2009). https://doi.org/10.1038/nprot.2009.98

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