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


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.

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We respectfully acknowledge the previously published work of Carlos Barbas' group, Sangamo Biosciences and ToolGen, Inc., which described and characterized the various zinc finger modules we used as the basis for our composite archive. We thank J.-S. Kim for providing the full amino acid sequences of the ToolGen human zinc finger modules. We thank members of our groups, especially P. Zaback and J. Townsend for helpful suggestions. J.D.S. is supported by USDA MGET 2001-52100-11506. A.S.H. was supported by NIH T32 CA09216. M.H.P is supported by the NIH (R01 HL0792595 and R21 CA120681). D.F.V. is supported by NSF grant DBI 0501678. J.K.J. is supported by the NIH (R01 GM069906 and R01 GM072621) and the MGH Department of Pathology. J.K.J. dedicates this protocol to the memory of Robert L. Burghoff, a patient teacher and friend who always knew how to make molecular biology experiments work. Note added in proof: Caroll, Segal and colleagues have recently described PCR-boned methods for assembling zinc finger modules into arrays and methods for purifying ZFNs and testing their activities in vitro (Caroll, D., Morton, J.J., Beumer, K.J. & Segal, D.J. Design, construction and in vitro testing of zinc finger nucleones. Nat. Protocols 3, 1329–1341).

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The authors declare no competing financial interests.

Correspondence to J Keith Joung.

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Figure 1: Schematic of (a) a zinc finger nuclease and (b) a zinc finger nuclease dimer bound to its target cleavage site.
Figure 2: Schematic of the bacterial two-hybrid reporter system.
Figure 3: Overview of restriction digest-based modular assembly.
Figure 4: Strategy for constructing B2H expression vectors and transformation of B2H reporter strains.
Figure 5: Strategy for constructing B2H reporter vectors.
Figure 6: Strategy for constructing plant or human ZFN expression vectors.


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