Engineering Designer Nucleases with Customized Cleavage Specificities

Jeffry D. Sander1, Morgan L. Maeder2, J. Keith Joung2

1 Department of Pathology, Harvard Medical School, Boston, Massachusetts, 2 Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 12.13
DOI:  10.1002/0471142727.mb1213s96
Online Posting Date:  October, 2011
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Engineered designer nucleases can be used to efficiently modify genomic sequence in a wide variety of model organisms and cell types. Zinc finger nucleases (ZFNs), consisting of an engineered zinc finger array fused to a non‐specific cleavage domain, have been extensively used to modify a broad range of endogenous genes. Protocols for engineering ZFNs targeted to specific gene sequences of interest using the context‐dependent assembly (CoDA) method are described in this unit. Curr. Protoc. Mol. Biol. 96:12.13.1‐12.13.16. © 2011 by John Wiley & Sons, Inc.

Keywords: zinc finger nucleases; zinc finger proteins; context‐dependent assembly; protein engineering; DNA‐binding domains

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Table of Contents

  • Introduction
  • Basic Protocol 1: Identifying CoDA ZFN Targets and Synthesizing CoDA ZFN‐Encoding DNA
  • Support Protocol 1: Bacterial Two‐Hybrid (B2H) Assay to Quantify DNA‐Binding Activities of Zinc Finger Arrays
  • Basic Protocol 2: Construction of ZFN Expression Vectors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Identifying CoDA ZFN Targets and Synthesizing CoDA ZFN‐Encoding DNA

  • DNA sequence of genomic region to target with ZFNs
  • Computer with internet access

Support Protocol 1: Bacterial Two‐Hybrid (B2H) Assay to Quantify DNA‐Binding Activities of Zinc Finger Arrays

  • pGP‐FF plasmid (plasmid, restriction map, and full sequence available from Addgene;
  • XbaI restriction enzyme (NEB)
  • BamHI‐HF restriction enzyme (NEB)
  • QIAquick gel extraction kit (Qiagen)
  • Synthesized zinc finger plasmid (see protocol 1)
  • Quick ligation kit (NEB)
  • Chemically competent XL1‐Blue cells (Stratagene)
  • LB broth (Difco, cat. no. 244620)
  • LB agar plates (Difco, cat. no. 244520) supplemented with 100 µg/ml carbenicillin
  • Carbenicillin (Sigma, cat. no. T4625)
  • QIAprep spin miniprep kit (Qiagen)
  • pBAC‐LacZ plasmid (plasmid, restriction map, and full DNA sequence available from Addgene;
  • BsaI restriction enzyme (NEB)
  • Nuclease‐free water
  • Cloned Pfu polymerase and 10× reaction buffer (Stratagene)
  • dCTP nucleotide
  • 10× annealing buffer (see recipe)
  • Chemically competent bacterial strain (Transformax Epi300, Epicentre)
  • Chloramphenicol
  • Arabinose
  • Chemically competent bacterial strain KJBAC1 (Addgene)
  • Expand high‐fidelity PCR kit (Roche Applied Science)
  • 1 M MgCl 2 ( appendix 22)
  • Solution A with glycerol (see recipe)
  • pAC‐Kan‐alpha‐Gal4 plasmid (Addgene)
  • LB agar plates (unit 1.1) supplemented with 100 µg/ml carbenicillin, 12.5 µg/ml chloramphenicol, and 30 µg/ml kanamycin
  • Kanamycin
  • 10 mM ZnSO 4
  • 500 mM IPTG
  • Lysis master mix (see recipe)
  • Z‐buffer with β‐mercaptoethanol (see recipe)
  • 4 mg/ml ONPG
  • 200‐µl and 1.5‐ml microcentrifuge tubes
  • 37°C, 42°C, 50°C, and 72°C water baths
  • 37°C incubator with shaking
  • 95°C heating block
  • 50‐ml conical tubes, sterile
  • 96‐well microtiter plates
  • 2‐ml assay blocks (Corning)
  • Microtiter plate reader with temperature control option (e.g., Biorad model 680)
  • Additional reagents and equipment for PCR (unit 15.1), gel purification (unit 2.5 or )

Basic Protocol 2: Construction of ZFN Expression Vectors

  • Nuclease expression plasmids (see Table 12.13.2):
    • pST1374, MLM290 and MLM292, or MLM800 and MLM802
  • Synthesized zinc finger constructs (see protocol 1)
  • XbaI (NEB)
  • BamHI (NEB)
  • NotI (NEB)
  • QIAquick gel extraction kit (Qiagen)
  • Nuclease‐free water
  • Quick ligation kit (NEB)
  • Chemically competent bacterial strain XL‐1 Blue (recA1 endA1 gyrA96 thi‐1 hsdR17 supE44 relA1 lac [F' proAB lacIq lacZDM15 Tn10 (TetR)]; Stratagene, cat. no. 200249)
  • LB medium (Difco, cat. no. 244620)
  • LB agar (Difco, cat. no. 244520) plates supplemented with 100 µg/ml carbenicillin
  • Carbenicillin (Sigma, cat. no T4625; 50 mg/ml–1 stock solution in water)
  • QIAprep spin miniprep kit (Qiagen)
  • 37°C water bath
  • 37°C shaking incubator
  • Additional reagents and equipment for gel isolation (unit 2.6)
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Literature Cited

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   Cornu, T.I., Thibodeau‐Beganny, S., Guhl, E., Alwin, S., Eichtinger, M., Joung, J., and Cathomen, T. 2008. DNA‐binding specificity is a major determinant of the activity and toxicity of zinc‐finger nucleases. Mol. Ther. 16:352‐358.
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Key References
  Sander et al., 2011. See above.
  Describes the CoDA method and demonstrates the efficacy of ZFNs produced by this approach in plants and zebrafish.
Internet Resources
  Provides access to the ZiFiT software program for engineering zinc fingers using CoDA.
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