Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly

Journal name:
Nature Protocols
Volume:
1,
Pages:
1637–1652
Year published:
DOI:
doi:10.1038/nprot.2006.259
Published online

Abstract

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using “modular assembly” have been described, standardized reagents and protocols that permit rapid, cross-platform “mixing-and-matching” of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24–26 d.

At a glance

Figures

  1. Schematic of (a) a zinc finger nuclease and (b) a zinc finger nuclease dimer bound to its target cleavage site.
    Figure 1: Schematic of (a) a zinc finger nuclease and (b) a zinc finger nuclease dimer bound to its target cleavage site.

    Individual zinc finger domains are depicted as colored spheres with F1 representing the amino-terminal finger, F2 the middle finger and F3 the carboxy-terminal finger in a three-finger array. The FokI DNA cleavage domain is represented as a purple colored octagon. Note that the spacer sequence between the two 9-bp 'half-sites' can be five or six base pairs.

  2. Schematic of the bacterial two-hybrid reporter system.
    Figure 2: Schematic of the bacterial two-hybrid reporter system.

    Coexpression of the zinc finger-Gal11P and alpha-Gal4 hybrid proteins in a B2H reporter strain leads to activated expression of a lacZ reporter gene if the multi-finger domain interacts with a target DNA-binding site located just upstream of the promoter39. This activation is mediated by recruitment of RNA polymerase complexes (that have incorporated alpha-Gal4) to the promoter by promoter-bound zinc finger-Gal11P proteins via interaction of Gal11P and Gal4. A three-finger array is depicted as three colored spheres (as in Fig. 1) and Gal11P represents a fragment of the yeast Gal11P protein (aa 263–352)39. The alpha-Gal4 hybrid protein consists of a portion of the E. coli RNA polymerase alpha-subunit (aa 1–248, encompassing the amino-terminal domain and inter-domain linker) and a fragment of yeast Gal4 (aa 58–97)39.

  3. Overview of restriction digest-based modular assembly.
    Figure 3: Overview of restriction digest-based modular assembly.

    Plasmids pc3XB-F1, -F2 and -F3 encode individual hypothetical finger modules from the archive cloned into plasmid pc3XB. Each finger coding sequence is flanked on the 5′ end by unique XbaI and XmaI sites and on the 3′ end by unique AgeI, BsgI and BamHI sites. The configuration of unique flanking restriction sites in all pc3XB-based plasmids permits any two fingers (e.g., F1 and F2) to be joined together by ligating a finger F1-encoding vector backbone (linearized by digestion with AgeI and BamHI) to a finger F2-encoding fragment (released from the plasmid by digestion with XmaI and BamHI). The resulting plasmid encodes a two-finger (F1 followed by F2) array which again is flanked on the 5′ end by XbaI and XmaI and on the 3′ end by AgeI, BsgI and BamHI. (Note that ligation of compatible XmaI and AgeI overhangs destroys both sites.) A third finger (F3) can be added to the array by ligating an F1/F2-encoding AgeI/BamHI-digested vector backbone to a F3-encoding XmaI/BamHI-digested fragment. Note that plasmid maps are not to scale.

  4. Strategy for constructing B2H expression vectors and transformation of B2H reporter strains.
    Figure 4: Strategy for constructing B2H expression vectors and transformation of B2H reporter strains.

    Zinc finger arrays assembled in the pc3XB plasmid can be cloned directly into vectors designed to express zinc finger arrays as a Gal11P hybrid protein (for use in B2H assays) using the unique XbaI and BsgI restriction sites. To perform B2H assays, the resulting plasmid (pGP-FB-F1/F2/F3) is co-transformed with the pAC-KAN-alphaGal4 plasmid into a “B2H reporter strain” harboring reporter plasmid pBAC-ZFBS-lacZ (ZFBS = zinc finger binding site). Note that the pGP-FB-F1/F2/F3, the pAC-KAN-alphaGal4 and the pBAC-ZFBS-lacZ plasmids confer resistance to ampicillin, kanamycin and chloramphenicol, respectively. Note that plasmid maps are not to scale.

  5. Strategy for constructing B2H reporter vectors.
    Figure 5: Strategy for constructing B2H reporter vectors.

    The top half of the figure illustrates the design and annealing of oligonucleotides comprising the zinc finger binding site as described in Box 2. The bottom half of the figure illustrates digestion of the plasmid pBAC-lacZ with BsaI and generation of extended overhangs by treatment with Pfu polymerase in the presence of a single deoxynucleotide (dCTP). Treatment with Pfu under these conditions will generate the specific overhangs shown that are complementary to the overhangs of the annealed oligonucleotide cassette. Ligation of the vector backbone to the annealed oligonucleotides will generate the desired pBAC-ZFBS-lacZ reporter vector. Note that plasmid maps are not to scale.

  6. Strategy for constructing plant or human ZFN expression vectors.
    Figure 6: Strategy for constructing plant or human ZFN expression vectors.

    Zinc finger arrays tested in the B2H system can be directly cloned from a pGP-FB-F1/F2/F3 expression vector using the unique XbaI and BamHI sites into a vector designed to express ZFNs in either plant (pDW1775) or human (pST1374) cells. The epitope tag (tag) in the plant ZFN expression vector is AcV5 whereas it is FLAG in the human ZFN expression vector. The nuclear localization signal (NLS) in both vectors is the SV40 NLS. Restriction sites referred to in the text are shown. Note that plasmid maps are not to scale.

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Author information

Affiliations

  1. Department of Genetics, Development & Cell Biology, Iowa State University, 1035A Roy J. Carver Co-Lab, Ames, Iowa 50011, USA.

    • David A Wright,
    • Jeffry D Sander,
    • Ronnie J Winfrey,
    • Fengli Fu,
    • Drena Dobbs &
    • Daniel F Voytas
  2. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, USA.

    • Stacey Thibodeau-Beganny,
    • Andrew S Hirsh,
    • Magdalena Eichtinger &
    • J Keith Joung
  3. Interdepartmental Graduate Program in Bioinformatics & Computational Biology, Iowa State University, 2114 Molecular Biology Building, Ames, Iowa 50011, USA.

    • Jeffry D Sander,
    • Fengli Fu &
    • Drena Dobbs
  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Andrew S Hirsh,
    • Magdalena Eichtinger &
    • J Keith Joung
  5. Departments of Pediatrics & Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.

    • Matthew H Porteus

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The authors declare no competing financial interests.

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