PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Protoc. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2855653
NIHMSID: NIHMS186240

Modular System for the Construction of Zinc-Finger Libraries and Proteins

Abstract

Engineered zinc-finger transcriptional factors (ZF-TF) are powerful tools to modulate the expression of specific genes. Complex libraries of ZF-TF can be delivered into cells to scan the genome for genes responsible for a particular phenotype or to select the most effective ZF-TF to regulate an individual gene. In both cases, the construction of highly representative and unbiased libraries is critical. In this work we describe a novel, user-friendly, zinc-finger technology suitable for the creation of complex libraries and the construction of customized ZF-TFs. The novel technology described here simplifies the building of zinc-finger libraries, avoids PCR-introduced bias, and ensures equal representation of every module. We also describe the construction of a customized ZF-TF that can be transferred to a number of expression vectors. This protocol can be completed in 9 to 11 days.

Introduction

Zinc-finger (ZF) domains of the Cys2-His2 class are among the most common DNA-binding motifs found in eukaryotes. Each 30-amino acid domain contains a single amphipathic helix responsible for binding three base pairs of DNA through the formation of specific contacts 1,2. These domains can be covalently linked into multi-modular proteins to recognize longer DNA sequences. When fused to effector domains such as transcriptional activators and repressors 3-17, methylases 18-21, recombinases 22,23, transposases24, integrases 25, nucleases 26-36, or other domains 37-40, ZF domains can be used to direct transcription (up-regulation or down-regulation of specific genes), modify genes (targeted mutations, gene repair, epigenetic modification, or gene replacement), or as serve as novel diagnostic tools. Six-finger proteins specifically recognize 18 base pair DNA sequences, in theory, a long enough region to be unique in the human or any other genome 11,41. This specificity has been demonstrated in transgenic plants and human cells using array analysis 8,42,43. Zinc-finger directed proteins have potential applications for the study of gene expression and function in normal and disease processes as well as for gene therapy for treatment of cancer and genetic disorders.

Polydactyl Zinc-Finger Proteins and Libraries

We have developed a simple system for creating zinc-finger libraries and specific proteins based on three unique vectors containing all of the GNN 3, ANN 4, or CNN 5 domains (Fig. 1, Supplementary Fig. 1, and Supplementary Sequence Archive (SSA)). This system will enable researchers who are not well versed in zinc-finger technology to easily assemble libraries and designed polydactyl zinc-finger proteins. The availability of these tools will greatly enhance the ability of scientists in the areas of gene therapy and gene regulation to specifically target a genomic site of interest.

Figure 1
Plasmid map of the SuperZiF plasmids GNN, ANN, and CNN. Unique restriction sites were placed between each zinc-finger module to enable isolation of individual and subsets of zinc fingers. The zinc-finger sequence was synthesized by Blue Heron and cloned ...

Libraries of zinc-finger proteins have proven to be useful for selection of endogenous gene regulators 44-54. Zinc-finger domains designed to bind a specific target may do so in vitro, but may not be able to bind the genomic target in vivo. Factors such as secondary structure, chromatin structure, or binding of other proteins may prevent the designed zinc-finger from binding to its intended genomic target. Using a library frees the researcher from choosing a predefined target site within a promoter. When an appropriate selection system is used in conjunction with the library, the zinc-finger transcriptional regulator that binds to the optimal region in the promoter can be discovered 44,45,54. This strategy may also result in the identification of novel, indirect gene regulators that may be useful for pathway discovery. Libraries can also be used for selection of specific phenotype changes to identify genes involved in normal and/or disease processes 46,48,51,52. The libraries created with this protocol can also be used in bacterial 55,56, yeast 57,58, and cell-free 59,60 zinc-finger selection systems.

Polydactyl Zinc-Finger Construction Methods

Two protocols describing creation of polydactyl zinc-fingers have been published 61,62. The Wright et al. protocol 62 describes modular assembly of zinc-finger proteins from a set of 140 zinc-finger modules utilized by the Zinc Finger Consortium Modular Assembly Kit. Most of the zinc finger modules used in this protocol and others are actually derived from zinc finger modules published by the Barbas laboratory. The Carroll et al. protocol 61 describes a PCR-based method for constructing multi-finger proteins that are again larger derived from our reported domains. Both of these systems are good for constructing individual and designed zinc fingers. However, libraries created with these systems would probably not be representative of all possible zinc-finger domains (note: the published manuscripts do not claim that these systems can be used to make libraries). One of the potential pitfalls of the Wright et al. protocol is that each zinc-finger must be carefully mixed in equal molar quantities to ensure that the library is not biased and that numerous helices of redundant binding specificity are present. The Carroll et al. protocol utilizes PCR and thus mutations introduced due to polymerase fidelity and differential annealing of degenerate primers would bias libraries.

Maeder et al.63 have used OPEN (Oligomerized Pool ENgineering) to create pools of three-finger proteins that target a defined 9 bp site. These pools can then be combined to select zinc-finger proteins that bind a 9 bp target sequence in a bacterial two-hybrid system. This system was successful in selecting three-finger zinc-finger proteins that when fused to nucleases were functional in human and plant cells. However, this system hinges on the construction of hundreds of pools of zinc fingers and the selection of the correct pools. The pools are also limited to GNNs and some TNNs, which limits the available target sites.

The SuperZiF System

The system described in this protocol is ideal for creating zinc-finger libraries and designed polydactyl zinc-finger proteins. Three SuperZiF plasmids were synthesized in the pUCminusMCS vector (Blue Heron) containing all zinc-finger domains previously published and characterized; one SuperZiF plasmid each for the GNN3, ANN4, and CNN5 zinc-finger domains (Fig. 1, Supplementary Fig. 1, and SSA). For library assembly, XhoI and XmaI restriction sites are 5′ to each finger and AgeI and SpeI sites are 3′ to each finger (Fig. 2, Supplementary Fig. 1, and SSA). When creating zinc-finger libraries, the SuperZiF plasmids can be cut with XhoI and SpeI to release each finger's coding sequence in equal molar quantities, thereby reducing library bias. When assembled into polydactyl zinc-finger proteins, each finger from the SuperZiF plasmid is equally represented at each position. If more than one SuperZiF plasmid is used to generate individual fingers, then the diversity of the potential binding sites for the library is increased accordingly. The SuperZiF zinc-fingers were built on the Sp1C backbone64, which has shown enhanced stability, increased affinity, and better expression 10 than the Zif268 backbone 2. To minimize repeated sequences, which may cause problems with recombination, plasmid propagation, and sequencing, the nucleotide sequence for the Sp1C backbone was varied without affecting the amino acid sequence (see SSA).

Figure 2
Library cloning scheme. The SuperZiF vector is digested with XhoI and SpeI and ligated into pCSV (see Supplementary Figure 2 for a complete map and the Supplementary Sequenced Archive for the plasmid sequence). Next, the 1ZF library is cut with XmaI and ...

The SuperZiF system is also useful for engineering proteins to target specific DNA sequences. The Zinc Finger Tools 65 website (www.zincfingertools.org) can be used to identify potential zinc-finger target sites and to design polydactyl zinc-fingers that target those specific sites. A scoring function is incorporated to help the user choose which of the proposed zinc-finger proteins are likely to be the most specific for the selected DNA target. For constructing designed proteins, unique restriction sites were placed between each finger, enabling the isolation of any individual finger from the appropriate SuperZiF vector (Fig. 1). Individual fingers can also be isolated and saved during the first step of the library construction process for future use when assembling designed proteins.

First we describe the method used to create a standard six-finger library from a single SuperZiF plasmid (SuperZiFGNN). Next, we describe a modified version of that method to create a five-finger library with 12 GNN (no GNGs) and 15 ANN zinc-fingers, which covers more sequence space than a library where every fourth base is the same. This GNH/ANN five-finger library was successfully used to select zinc-fingers proteins that were able to bind the γ-globin promoter and up-regulate the transcription of this gene54. We have also included a method to create designed zinc-finger proteins from either the SuperZiF vectors or from preselected one-finger clones, which can easily be recovered from the first steps in the construction of a standard six-finger library. This method has been successfully used for the rapid assembly of chimeric zinc finger recombinases capable of transgene integration into the human genome with more than 98% accuracy 66.

Once the zinc-finger libraries or designed clones are constructed, they can be cloned into a variety of expression vectors using the SfiI restriction sites that flank the zinc-finger cassettes. Our group has constructed retroviral, lentiviral, and transient expression vectors with both transcriptional activators and repressors (Table 1, Supplementary Fig. 2 and SSA) designed for SfiI cloning of polydactyl zinc-fingers which are available upon request10,11,54,67. An adapted pMAL-c vector (New England Biolabs) 41 is also available for bacterial expression and purification via maltose-binding protein fusion. Using these vectors, selection methods can be employed to isolate zinc-finger transcription factors with the desired target specificity44,45,54 or phenotype change46,48,51,52.

Materials

Reagents

  • SuperZifGNN (Blue Heron, see Supplementary Fig. 1 and SSA)
  • SuperZifANN (Blue Heron, see Supplementary Fig. 1 and SSA)
  • SuperZifCNN (Blue Heron, see Supplementary Fig. 1 and SSA)
  • Expression vectors (see Table 1) (see Supplementary Fig. 2 and SSA)
  • Bacteria strain SS320 (F lacI22 lacZ pro-48 met-90 trpA trpR his-85 rpsL azi-9 gyrA λ P1s; a gift from Dr. Sachdev S. Sidhu at Genentech68. Electrocompetent SS320 cells are available from Lucigen, 60512-1)
  • Bacteria strain XL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F'proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, 200228)
  • Restriction enzymes (from New England Biolabs): XhoI (R0146L), Spe(R0133L)I, SfiI(R0123L) (20 U/μL), AgeI (R0552L), XmaI(R0180L), NheI(R0131L), AccI(R0161S), AflII(R05205)
  • Restriction enzymes (from Roche): EcoRI (1175084), SfiI(1288059) (40 U/μL)
  • 10× restriction enzyme buffers (New England Biolabs)
  • T4 DNA Ligase and T4 DNA Ligase buffers (Invitrogen, 15224090)
  • Calf intestinal phosphatase (CIP, New England Biolabs, M0290L))
  • PureLink PCR Purification kit (Invitrogen, K310002)
  • QIAquick Gel Extraction kit (Qiagen, 28704)
  • Ultrafree-MC centrifuge filter units, 0.4 μm (Millipore, UFC30HVNB) (For “Freeze and Squeeze”69)
  • HiPure Plasmid Filter Maxiprep kit (Invitrogen, K210017)
  • pSCVseqF: 5′-GTA AAA CGA CGG CCA GTG AGC GC-3′
  • pSCVseqB: 5′- GAT ACC GCT CGC CGC AGC CGA AC-3′
  • 100% ethanol
  • 3 M Sodium acetate
  • Glycogen (Roche, 901393)
  • SB medium (see Reagent Setup)
  • SOC medium (see Reagent Setup)
  • Carbenicillin (Omega Scientific)
  • UltraPure agarose (Invitrogen, 16500500)
  • Tris-acetate EDTA (TAE)
  • Ethidium Bromide 10 mg/mL solution (EtBr) (Sigma-Aldrich, E1510) CAUTION – carcinogen, use proper personal protective equipment including gloves.

Equipment

  • Electroporation cuvettes, 2 mm gap (VWR, 89047-208)
  • Electroporator (BioRad)
  • Sterile microcentrifuge tubes
  • Sterile 14-mL culture tubes (Falcon)
  • Standard orbital shaker for growing bacterial cultures
  • Heating block or water bath for enzyme digestions
  • Sterile 250 mL centrifuge bottles
  • Electrophoresis system (Fisher Biotech)

Reagent Setup

  • SB medium (1 L):
    • 10 g MOPS (hemisodium salt)
    • 30 g Tryptone Peptone
    • 20 g Yeast Extract
    • Dissolve in dH2O, adjust volume, autoclave
  • Store at 4°C for up to 6 months
  • SOC medium (1 L):
    • 20 g Tryptone peptone
    • 5 g Yeast extract
    • 0.5 g NaCl
    • Dissolve in 800 mL dH20
    • Add 10 mL of 0.25 M KCl
    • Adjust pH to 7.0
    • Add 10 mL of 1 M MgCl2
    • Adjust volume to 1 L and autoclave
    • Add 1 mL of 1 M glucose
  • Store at 4°C for up to 6 months

The SuperZiF, pSCV, and expression plasmids listed in Table 1 are available from our laboratory upon request.

Procedure

Construction of a six-finger GNN library. Timing: 9 days

1| Create a one-finger library from the SuperZiFGNN plasmid. Digest 10 μg SuperZiFGNN with XhoI and SpeI (shown below) at 37°C for 4 hours to create a 1ZF insert.


ComponentAmount

SuperZifGNN10 μg
XhoI (20 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 2)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Also digest 5 μg pSCV with XhoI and SpeI (shown below) at 37°C for 4 hours. Once this digestion is complete, add 1 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

pSCV5 μg
XhoI (20 U/μL)1 μL
SpeI (10 U/μL)1 μL
10× Buffer (NEB 2)2.5 μL
10× BSA2.5 μL

Nuclease-free waterto 25 μL

Total25 μL

2| Purify the digested SuperZiFGNN DNA fragment by electrophoresis on 2% agarose gel (1× TAE with EtBr). Cut out the 100-bp band and purify by “Freeze and Squeeze”69 or with the QIAquick Gel Extraction kit.

Purify the pSCV vector with the PureLink PCR Purification kit (Invitrogen).

PAUSE POINT Purified vector and insert can be stored at -20°C indefinitely.

3| Ligate purified pSCV vector and the 1ZF GNN insert using T4 DNA ligase (shown below). Also set up a control ligation with no insert to assess the library background (i.e., efficiency of digestion and dephosphorylation). Allow the ligation reaction to proceed overnight at room temperature (approximately 25°C).


ComponentLigationControl

pSCV vector100 ng100 ng
1ZF GNN insert50 ng-------
5× T4 DNA Ligase Buffer4 μL4 μL
T4 DNA Ligase (1 U/μL)1 μL1 μL
Nuclease-free waterto 20 μLto 20 μL

Total20 μL20 μL

4| Ethanol precipitate the ligation reactions. Add 2 μL 3 M sodium acetate (NaOAc, pH ~5.2-6.0), 1 μL glycogen (1 mg/mL) (optional), and 50 μL of absolute ethanol. Let stand at -20°C for at least 1 hour. Centrifuge at ≥ 15,000 xg for 30 minutes. Remove supernatant and wash with 500 μL 70% ethanol. Centrifuge at ≥ 15,000 xg for 15 minutes. Remove supernatant and allow pellet to air-dry for 15 minutes. Resuspend DNA pellet in 10 μL nuclease-free water.

PAUSE POINT Ligations can be stored in the NaOAc/ethanol solution or after resuspension in water at -20°C indefinitely.

5| Transform 5 μL each of the ligation and control in 75 μL competent cells via electroporation in a 2-mm gap cuvette (2.5 kV, 15.0 μF, 200 Ohms). We recommend SS32068 cells due to high competency needed for later stages, however at this stage, XL1Blue cells are acceptable.

6| After electroporation, add 1 mL SOC media each to the transformed ligation and control and transfer to 14-mL culture tubes. Incubate with agitation (250 rpm) at 37°C for 1 hour.

7| Plate 0.1 μL and 1 μL of the transformed ligation and 1 μL and 10 μL of the transformed control onto SB plates containing 100 μg/mL carbenicillin. Allow these to grow overnight at 37°C. Mix the remaining transformed ligation with 100 mL SB with 50 μg/mL carbenicillin. Incubate the culture with agitation (250 rpm) overnight at 37°C.

8| Count colonies on the transformed ligation and control plates. To ensure 10× coverage of the library 160 colonies per 1000 μL of SOC culture are required. The transformed ligation plates should have at least 10-fold more colonies than the control plates to ensure less than 10% background.

?TROUBLESHOOTING

9| Do a diagnostic digest for quality assurance of pSCV-1ZFGNN. Use the HiPure Plasmid Filter Maxiprep kit (Invitrogen) to recover the library of plasmid DNA from the 100 mL culture. Digest the library with SfiI for 1 hour at 50°C (shown below) to ensure the insert is the correct size. After digestion use electrophoresis on a 2% agarose TAE gel with EtBr to ensure that a 100-bp band fragment is released. We occasionally see multiple inserts (a 300 bp fragment released) at this step. Please see the troubleshooting guide for solutions to this problem.


ComponentAmount

pSCV-1ZFGNN100 ng
SfiI (20 U/μL)0.5 μL
10× Buffer (NEB 2)2 μL
10× BSA2 μL
Nuclease-free waterto 20 μL

Total20 μL

?TROUBLESHOOTING

PAUSE POINT Purified plasmid DNA can be stored at -20°C indefinitely.

10| (optional) It may be helpful to pick, miniprep, and sequence individual clones from the 1ZF library. This step ensures that the library is diverse and not contaminated by a single clone. These clones can also be saved and used for cloning designed multi-finger proteins.

Pick 20 colonies from the transformed ligation plates and inoculate 2.5 mL SB media containing 50 μg/mL carbenicillin. Shake (250 rpm) overnight at 37°C.

Prepare small-scale plasmid purifications (PureLink Quick Plasmid Miniprep kit from Invitrogen) from the overnight cultures and sequence with pSCVseqF primer.

PAUSE POINT Purified plasmid DNA can be stored at -20°C indefinitely

11| Create 2ZF GNN library. Digest SuperZifGNN with XmaI and SpeI (shown below) at 37°C for 4 hours to create a 1ZF insert.


ComponentAmount

SuperZifGNN10 μg
XmaI (10 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Also digest 5 μg pSCV-1ZFGNN with AgeI and SpeI (shown below) at 37°C for 4 hours to create a 1ZF vector. Once this digestion is complete, add 1 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

pSCV-1ZFGNN5 μg
AgeI (5 U/μL)1 μL
SpeI (10 U/μL)1 μL
10× Buffer (NEB 4)2.5 μL
10× BSA2.5 μL
Nuclease-free waterto 25 μL

Total25 μL

12| Repeat step 2 to purify the insert and vector. The insert fragment should be 100 bp.

PAUSE POINT Purified vector and insert can be stored at -20°C indefinitely.

13| Ligate purified 1ZF GNN vector and the 1ZF GNN insert using T4 DNA ligase (shown below). Also set up a control ligation with no insert to assess the library background. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLigationControl

1ZF GNN vector100 ng100 ng
1ZF GNN insert50 ng-------
5× T4 DNA Ligase Buffer4 μL4 μL
T4 DNA Ligase1 μL1 μL
Nuclease-free waterto 20 μLto 20 μL

Total20 μL20 μL

14| Repeat steps 4 though 7.

15| Count colonies on the transformed ligation and control plates. To ensure 10× coverage of the library 2560 (162×10) colonies per 1000 μL of SOC culture are required. The transformed ligation plates should have at least 10-fold more colonies than the control plates to ensure less than 10% background.

?TROUBLESHOOTING

16| Repeat steps 9 and 10. The fragment released from the library should be 200 bp. We occasionally see multiple inserts (a 400 bp fragment released) at this step. Please see the troubleshooting guide for solutions to this problem.

?TROUBLESHOOTING

17| Create 3ZF GNN library. Digest 5 μg pSCV-2ZFGNN with AgeI and SpeI (shown below) at 37°C for 4 hours to create a 2ZF vector. Once this digestion is complete, add 1 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

pSCV-2ZFGNN5 μg
AgeI (5 U/μL)1 μL
SpeI (10 U/μL)1 μL
10× Buffer (NEB 4)2.5 μL
10× BSA2.5 μL
Nuclease-free waterto 25 μL

Total25 μL

18| Purify the pSCV-2ZFGNN vector with the PureLink PCR Purification kit (Invitrogen).

PAUSE POINT Purified vector can be stored at -20°C indefinitely.

19| Ligate purified 2ZF GNN vector and the 1ZF GNN insert (from step 11) using T4 DNA ligase (shown below). Also set up a control ligation with no insert to assess the library background. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLigationControl

2ZF GNN vector200 ng200 ng
1ZF GNN insert100 ng-------
5× T4 DNA Ligase Buffer8 μL8 μL
T4 DNA Ligase2 μL2 μL
Nuclease-free waterto 40 μLto 40 μL

Total40 μL40 μL

20| Ethanol precipitate the ligation reactions. Add 4 μL 3 M sodium acetate (NaOAc, pH ~5.2-6.0), 1 μL glycogen (1 mg/mL) (optional), and 100 μL of absolute ethanol. Let stand at -20°C for at least 1 hour. Centrifuge at ≥ 15,000 xg for 30 minutes. Remove supernatant and wash with 500 μL 70% ethanol. Centrifuge at ≥ 15,000 xg for 15 minutes. Remove supernatant and allow pellet to air-dry for 15 minutes. Resuspend DNA pellet in 10 μL nuclease-free water.

PAUSE POINT Ligations can be stored in the NaOAc/ethanol solution or after resuspension in water at -20°C indefinitely.

21| Add 10 μL of the ligation mixture into 200 μL of competent cells and split into two cuvettes; transform via electroporation (2.5 kV, 15.0 μF, 200 Ohms). Transform 5 μL of the control in 100 μL competent cells via electroporation (2.5 kV, 15.0 μF, 200 Ohms). We recommend SS32068 cells due to their high competency.

22| After electroporation, add 1 mL SOC media each to the transformed ligation and control and transfer to 15 mL culture tubes combining the two transformed ligation cuvettes. Incubate with agitation (250 rpm) at 37°C for 1 hour.

23| Make a 1:100 dilution of the transformed ligation culture in SOC media. Plate 2 μL and 20 μL of the diluted transformed ligation culture and the transformed control culture onto SB plates containing 100 μg/mL carbenicillin. Allow these to grow overnight at 37°C. Mix the remaining transformed ligation with 100 mL SB with 50 μg/mL carbenicillin. Incubate the culture with agitation (250 rpm) overnight at 37°C.

24| Count colonies on the transformed ligation and control plates. To ensure 10× coverage of the library 40,960 (163×10) colonies per 2000 μL of SOC culture are required. The transformed ligation plates should have at least 20-fold more colonies than the control plates to ensure less than 10% background.

?TROUBLESHOOTING

25| Repeat steps 9 and 10. The fragment released from the library should be 300 bp. We occasionally see multiple inserts (a 500 bp fragment released) at this step. Please see the troubleshooting guide for solutions to this problem.

?TROUBLESHOOTING

26| Create 6ZF GNN library. Digest pSCV-3ZFGNN with XmaI and SpeI (shown below) at 37°C for 4 hours to create a 3ZF insert.


ComponentAmount

pSCV-3ZFGNN10 μg
XmaI (10 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Also digest 10 μg pSCV-3ZFGNN with AgeI and SpeI (shown below) at 37°C for 4 hours to create a 3ZF vector. Once this digestion is complete, add 2 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

pSCV-3ZFGNH/ANN10 μg
AgeI (5 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

27| Repeat step 2 to purify the insert and vector. The insert fragment should be 300 bp.

PAUSE POINT Purified vector and insert can be stored at -20°C indefinitely.

28| Ligate purified 3ZF GNN vector and 3ZF GNN insert using T4 DNA ligase (shown below). This master mix should be divided into three microcentrifuge tubes. Also set up a control ligation with no insert to assess the library background. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLigationControl

3ZF GNN vector600 ng200 ng
3ZF GNN insert900 ng-------
5× T4 DNA Ligase Buffer48 μL16 μL
T4 DNA Ligase12 μL4 μL
Nuclease-free waterto 240 μLto 80 μL

Total240 μL80 μL

29| Ethanol precipitate the ligation and control reactions. Add 8 μL 3 M sodium acetate (NaOAc, pH ~5.2-6.0), 1 μL glycogen (1 mg/mL) (optional), and 200 μL of absolute ethanol. Let stand at -20°C for at least 1 hour. Centrifuge at ≥ 15,000 xg for 30 minutes. Remove supernatant and wash with 1 mL 70% ethanol. Centrifuge at ≥ 15,000 xg for 15 minutes. Remove supernatant and allow pellet to air-dry for 15 minutes. Resuspend DNA pellet in 10 μL nuclease-free water.

PAUSE POINT Ligations can be stored in the NaOAc/ethanol solution or after resuspension in water at -20°C indefinitely.

30| Transform all 30 μL of the three ligation reactions (5 μL + 100 μL of competent cells × 6 cuvettes) and 5 μL of the control in 100 μL competent cells via electroporation (2.5 kV, 15.0 μF, 200 Ohms). We recommend SS32068 cells due to their high competency.

31| After electroporation, add 1 mL SOC media each to the transformed ligations and control and transfer to 14-mL culture tubes pooling two transformed ligation cuvettes into one culture tube (three ligation tubes and one control culture tube). Incubate with agitation (250 rpm) at 37°C for 1 hour.

32| Combine the three ligation cultures into one tube. Make a 1:100 dilution of the ligation culture in SOC media. Plate 0.6 and 6 μL of the diluted ligation culture and the control culture onto SB plates containing 100 μg/mL carbenicillin. Allow these to grow overnight at 37°C. Split the remaining transformed ligation culture into two 150-mL flasks of SB with 50 μg/mL carbenicillin. Incubate the culture with agitation (250 rpm) overnight at 37°C.

33| Count colonies on the transformed ligation and control plates. To ensure 10× coverage of the library 1.7×108 (166×10) colonies per 6000 μL of SOC culture are required. The transformed ligation plates should have at least 60-fold more colonies than the control plates to ensure less than 10% background.

?TROUBLESHOOTING

34| Repeat steps 9 and 10. The fragment released from the library should be 600 bp.

?TROUBLESHOOTING

Construction of a five-finger GNH/ANN library. Timing: 11 days

35| Create a one finger GNH (H= A, T, or C) library. Digest 10 μg of the SuperZifGNN plasmid with NheI and EcoRI (shown below) at 37°C for 4 hours to remove the GNGs from the vector.


ComponentAmount

SuperZifGNN10 μg
NheI (10 U/μL)1.5 μL
EcoRI (20 U/μL)1.5 μL
10× Buffer (NEB 2)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

36| Purify the digested SuperZiFGNH DNA fragment by electrophoresis on 1.5% agarose gel (1× TAE with EtBr). Cut out the ~4550-bp band and purify by “Freeze and Squeeze”69 or with the QIAquick Gel Extraction kit.

PAUSE POINT Purified insert can be stored at -20°C indefinitely.

37| Digest the SuperZifGNH fragment with XhoI and SpeI (shown below) at 37°C for 4 hours to create a 1ZFGNH insert.


ComponentAmount

SuperZifGNN2.5 μg
XhoI (20 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 2)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

38| Purify the digested GNH DNA fragment by electrophoresis on 2% agarose gel (1× TAE with EtBr). Cut out the 100-bp band and purify by “Freeze and Squeeze”69 or with the QIAquick Gel Extraction kit.

39| Digest, CIP, and purify the pSCV vector as instructed in steps 1 and 2.

PAUSE POINT Purified vector and insert can be stored at -20°C indefinitely.

40| Ligate the GNH insert and pSCV vector as instructed in step 3.

41| Perform steps 4 through 7 with the pSCV-GNH ligation.

42| Count colonies on the transformed ligation and control plates. To ensure 10× coverage of the library 120 colonies per 1000 μL of SOC culture are required. The transformed ligation plates should have at least 10-fold more colonies than the control plates to ensure less than 10% background.

?TROUBLESHOOTING

43| Perform steps 9 and 10 with pSCV-1ZFGNH. We occasionally see multiple inserts (a 300 bp fragment released) at this step. Please see the troubleshooting guide for solutions to this problem. We highly recommend sequencing several clones from this library to check for possible GNG contamination.

?TROUBLESHOOTING

44| Create a one finger GNH/ANN library. Digest 10 μg of pSCV-1ZFGNH and 10 μg of SuperZiFANN with XhoI and SpeI (shown below) at 37°C for 4 hours to create 1ZFGNH and 1ZFANN inserts.


ComponentAmount

pSCV-1ZFGNH or SuperZifANN10 μg
XhoI (20 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 2)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

45| Purify the digested GNH and ANN DNA fragments by electrophoresis on 2% agarose gel (1× TAE with EtBr). Cut out the 100-bp band and purify by “Freeze and Squeeze”69 or with the QIAquick Gel Extraction kit.

PAUSE POINT Purified inserts can be stored at -20°C indefinitely.

46| Ligate purified pSCV vector (from step 38) and the 1ZF GNH and ANN inserts using T4 DNA ligase (shown below). To ensure that each zinc-finger is present in equal amounts, the ratio of GNH to ANN should be 4:5. Also set up a control ligation with no inserts to assess the library background. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLigationControl

pSCV vector100 ng100 ng
1ZF GNH insert22.2 ng-------
1ZF ANN insert27.8 ng-------
5× T4 DNA Ligase Buffer4 μL4 μL
T4 DNA Ligase (1 U/μL)1 μL1 μL
Nuclease-free waterto 20 μLto 20 μL

Total20 μL20 μL

47| Perform steps 4 through 7 with the pSCV-GNH/ANN ligation.

48| Count colonies on the transformed ligation and control plates. To ensure 10× coverage of the library 270 colonies per 1000 μL of SOC culture are required. The transformed ligation plates should have at least 10-fold more colonies than the control plates to ensure less than 10% background.

?TROUBLESHOOTING

49| Perform steps 9 and 10 with pSCV-1ZFGNH/ANN. We occasionally see multiple inserts (a 400 bp fragment released) at this step. Please see the troubleshooting guide for solutions to this problem.

?TROUBLESHOOTING

50| Proceed as described in steps 11 though 25 to make pSCV-3ZFGNH/ANN, except at step 11 use the pSCV-1ZFGNH/ANN library to make the 1ZFGNH/ANN insert. The 2ZF library should have 7290 (272×10) colonies per 1000 μL of SOC culture and the 3ZF library should have 1.96×105 (273×10) colonies per 2000 μL of SOC culture.

?TROUBLESHOOTING

51| Create a 5ZF GNH/ANN library. Digest pSCV-2ZFGNHANN with XmaI and SpeI (shown below) at 37°C for 4 hours to create a 2ZF insert.


ComponentAmount

pSCV-2ZFGNH/ANN10 μg
XmaI (10 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Also digest 10 μg pSCV-3ZFGNH/ANN with AgeI and SpeI (shown below) at 37°C for 4 hours to create a 3ZF vector. Once this digestion is complete, add 2 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

pSCV-3ZFGNN10 μg
AgeI (5 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

52| Repeat step 2 to purify the insert and vector. The insert fragment should be 200 bp.

PAUSE POINT Purified vector and insert can be stored at -20°C indefinitely.

53| Ligate purified 3ZF GNH/ANN vector and 2ZF GNH/ANN insert using T4 DNA ligase (shown below). This master mix should be divided into three microcentrifuge tubes. Also, set up a control ligation with no insert to assess the library background. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLigationControl

3ZF GNH/ANN vector600 ng200 ng
2ZF GNH/ANN insert900 ng-------
5× T4 DNA Ligase Buffer48 μL16 μL
T4 DNA Ligase12 μL4 μL
Nuclease-free waterto 240 μLto 80 μL

Total240 μL80 μL

54| Perform steps 29 through 34 to complete the construction of pSCV-5ZFGNH/ANN. To ensure 10× coverage of the library 1.4×108 (275×10) colonies per 6000 μL of SOC culture are needed. The transformed ligation plates should have at least 60-fold more colonies than the control plates to ensure less than 10% background. It is not feasible to make a 6ZF-GNH/ANN library due to limitations of transformation efficiency (276×10 = 3.9×109).

?TROUBLESHOOTING

Construction of a designed six-finger clone (for example, recognizing 5′-GAA GAA GAA GAA GAA GAA-3′) Timing: 13 days

55| Use Zinc Finger Tools website to design a polydactyl zinc-finger.

56| Finger 1 cloning: Digest 10 μg SuperZiFGNN with unique enzymes that release the desire first zinc finger. As an example, use AccI and AflII to cut out the GAA-specific helix and digest (shown below) at 37°C for 4 hours to create the ZF1 insert.


ComponentAmount

SuperZifGNN10 μg
AccI (10 U/μL)1.5 μL
AflII (20 U/μL)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Also digest 5 μg pSCV with XhoI and SpeI (shown below) at 37°C for 4 hours. Once this digestion is complete, add 1 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

pSCV5 μg
XhoI (20 U/μL)1 μL
SpeI (10 U/μL)1 μL
10× Buffer (NEB 2)2.5 μL
10× BSA2.5 μL
Nuclease-free waterto 25 μL

Total25 μL

57| Purify the digested ZF1 DNA fragment by electrophoresis on 2% agarose gel (1× TAE with EtBr). Cut out the 100-bp band and purify by “Freeze and Squeeze”69 or with the QIAquick Gel Extraction kit.

Purify the pSCV vector with the PureLink PCR Purification kit (Invitrogen).

PAUSE POINT Purified vector and insert can be stored at -20°C indefinitely.

58| Digest the purified ZF1 fragment with XhoI and SpeI (shown below) at 37°C for 4 hours to create a compatible ZF1 insert.


ComponentAmount

Purified ZF12.5 μg
XhoI (20 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 2)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Purify the XhoI/ SpeI ZF1 insert with the PureLink PCR Purification kit (Invitrogen).

PAUSE POINT Purified insert can be stored at -20°C indefinitely.

59| Ligate purified pSCV vector and the ZF1-compatible insert using T4 DNA ligase (shown below). Also set up a control ligation with no insert to assess the ligation background. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLigationControl

pSCV vector100 ng100 ng
ZF1 insert50 ng-------
5× T4 DNA Ligase Buffer4 μL4 μL
T4 DNA Ligase (1 U/μL)1 μL1 μL
Nuclease-free waterto 20 μLto 20 μL

Total20 μL20 μL

60 Ethanol precipitate the ligation reactions. Add 2 μL of 3 M sodium acetate (NaOAc, pH ~5.2-6.0), 1 μL glycogen (1 mg/mL) (optional), and 50 μL of absolute ethanol. Let stand at −20°C for at least 1 hour. Centrifuge at ≥ 15,000 xg for 30 minutes. Remove supernatant and wash with 500 μL 70% ethanol. Centrifuge at ≥ 15,000 xg for 15 minutes. Remove supernatant and allow pellet to air-dry for 15 minutes. Resuspend DNA pellet in 10 μL nuclease-free water.

PAUSE POINT Ligations can be stored in the NaOAc/ethanol solution or after resuspension in water at -20°C indefinitely.

61| Transform 5 μL each of the ligation and control in 75 μL XL1-Blue competent cells via electroporation in a 2-mm gap cuvette (2.5 kV, 15.0 μF, 200 Ohms).

62| After electroporation, add 1 mL SOC media each to the transformed ligation and control and transfer to 15-mL culture tubes. Incubate with agitation (250 rpm) at 37°C for 1 hour.

63| Plate 1 μL and 10 μL of both the transformed ligation and control onto SB plates containing 100 μg/mL carbenicillin. Allow these to grow overnight at 37°C.

64| Pick several colonies, miniprep, and SfiI digest for 1 hour at 50°C (shown below) to ensure the insert is the correct size. Clones that have ZF1 inserted should release a 100-bp band and will become the ZF1 clone. Clones can be checked by sequencing with pSCVseqF primer.


ComponentAmount

pSCV-ZF1100 ng
SfiI (20 U/μL)0.5 μL
10× Buffer (NEB 2)2 μL
10× BSA2 μL
Nuclease-free water13.5 μL

Total20 μL

Optional: Individual clones from a one-finger library can be used as ZF1.

PAUSE POINT Purified plasmid DNA can be stored at -20°C indefinitely.

?TROUBLESHOOTING

65| Create ZF1-2 clone. Digest SuperZiFGNN plasmid with unique enzymes that release the desired second zinc finger domain. As an example, use AccI and AflII to cut out GAA specific helix and digest (shown below) at 37°C for 4 hours to create a ZF2 insert.


ComponentAmount

SuperZifGNN10 μg
AccI (10 U/μL)1.5 μL
AflII (20 U/μL)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Also digest 5 μg pSCV-ZF1 with AgeI and SpeI (shown below) at 37°C for 4 hours to create a pSCV-ZF1 vector. Once this digestion is complete, add 1 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

pSCV-ZF15 μg
AgeI (5 U/μL)1 μL
SpeI (10 U/μL)1 μL
10× Buffer (NEB 4)2.5 μL
10× BSA2.5 μL
Nuclease-free waterto 25 μL

Total25 μL

Purify the pSCV-ZF1 vector with the PureLink PCR Purification kit (Invitrogen).

PAUSE POINT Purified vector and insert can be stored at -20°C indefinitely.

66| Digest the purified ZF2 fragment with XmaI and SpeI (shown below) at 37°C for 4 hours to create a compatible ZF2 insert.


ComponentAmount

Purified ZF22.5 μg
XmaI (10 U/μL)1.5 μL
SpeI (10 U/μl)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Purify the XmaI/ SpeI ZF2 insert with the PureLink PCR Purification kit (Invitrogen).

PAUSE POINT Purified insert can be stored at -20°C indefinitely.

67| Ligate purified pSCV-ZF1 vector and the ZF2 insert using T4 DNA ligase (shown below). Also set up a control ligation with no insert to assess background. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLigationControl

pSCV-ZF1 vector100 ng100 ng
ZF2 insert50 ng-------
5× T4 DNA Ligase Buffer4 μL4 μL
T4 DNA Ligase1 μL1 μL
Nuclease-free waterto 20 μLto 20 μL

Total20 μL20 μL

68| Repeat steps 60 though 64 to obtain a clone that contains ZF1-2. In this case the released band after a SfiI digestion should be 200 bp.

?TROUBLESHOOTING

69| Create ZF1-2-3 clone. Digest SuperZiFGNN plasmid with unique enzymes that release the desire zinc finger. As an example, use AccI and AflII to cut out GAA specific helix and digest (shown below) at 37°C for 4 hours to create a ZF3 insert.


ComponentAmount

SuperZifGNN10 μg
AccI (10 U/μL)1.5 μL
AflII (20 U/μL)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Also digest 5 μg pSCV-ZF1-2 with AgeI and SpeI (shown below) at 37°C for 4 hours to create a ZF1-2 vector. Once this digestion is complete, add 1 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

pSCV-ZF1-25 μg
AgeI (5 U/μL)1 μL
SpeI (10 U/μL)1 μL
10× Buffer (NEB 4)2.5 μL
10× BSA2.5 μL
Nuclease-free waterto 25 μL

Total25 μL

70| Repeat digestion and purification described in step 66 to generate a compatible ZF3 insert.

PAUSE POINT Purified insert can be stored at -20°C indefinitely.

71| Ligate purified pSCV-ZF1-2 vector and the ZF3 insert using T4 DNA ligase (shown below). Also set up a control ligation with no insert to assess background. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLigationControl

pSCV-ZF1-2 vector100 ng100 ng
ZF3 insert50 ng-------
5× T4 DNA Ligase Buffer4 μL4 μL
T4 DNA Ligase1 μL1 μL
Nuclease-free waterto 20 μLto 20 μL

Total20 μL20 μL

72| Repeat steps 60 though 64 to obtain a clone that contains ZF1-2-3. In this case the released band after a SfiI digestion should be 300 bp.

?TROUBLESHOOTING

73| Create pSCV-ZF4-5-6 by repeating steps 56 through 72 using the appropriate zinc fingers for positions 4, 5, and 6.

74| Create pSCV-ZF1-2-3-4-5-6 clone. Digest pSCV-ZF4-5-6 plasmid with XmaI and SpeI (shown below) at 37°C for 4 hours to create a ZF4-5-6 insert.


ComponentAmount

pSCV-ZF4-5-610 μg
XmaI (10 U/μL)1.5 μL
SpeI (10 U/μL)1.5 μL
10× Buffer (NEB 4)5 μL
10× BSA5 μL
Nuclease-free waterto 50 μL

Total50 μL

Also digest 5 μg pSCV-ZF1-2-3 with AgeI and SpeI (shown below) at 37°C for 4 hours. Once this digestion is complete, add 1 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

pSCV-ZF1-2-35 μg
AgeI (5 U/μL)1 μL
SpeI (10 U/μL)1 μL
10× Buffer (NEB 4)2.5 μL
10× BSA2.5 μL
Nuclease-free waterto 25 μL

Total25 μL

75| Purify the digested ZF4-5-6 DNA fragment by electrophoresis on 2% agarose gel (1× TAE with EtBr). Cut out the 300-bp band and purify by “Freeze and Squeeze”69 or with the QIAquick Gel Extraction kit.

Purify the pSCV-ZF4-5-6 vector with the PureLink PCR Purification kit (Invitrogen).

PAUSE POINT Purified vector and insert can be stored at -20°C indefinitely.

76| Ligate purified pSCV-ZF1-2-3 vector and the purified ZF4-5-6 insert using T4 DNA ligase (shown below). Also set up a control ligation with no insert to assess background. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLigationControl

ZF1-2-3 vector100 ng100 ng
ZF4-5-6 insert50 ng-------
5× T4 DNA Ligase Buffer4 μL4 μL
T4 DNA Ligase1 μL1 μL
Nuclease-free waterto 20 μLto 20 μL

Total20 μL20 μL

77| Repeat steps 60 though 64 to obtain a 6-finger clone. In this case the released band after a SfiI digestion should be 600 bp.

?TROUBLESHOOTING

Transferring a zinc-finger library or protein to an expression vector. Timing: 3 days

78| Digest pSCV-Library/Clone with SfiI (shown below) at 50°C for 5 hours to create a Library or Clone insert.


ComponentLibraryClone

pSCV-Library/Clone10 μg5 μg
SfiI (40 U/μL)1.5 μL1 μL
10× Buffer (Roche M)5 μL2.5 μL
Nuclease-free waterto 50 μLto 25 μL

Total50 μL25 μL

Also digest the target expression vector (See Table 1) with SfiI (shown below) at 50°C for 5 hours. Once this digestion is complete, add 1 U calf intestinal phosphatase (CIP) and incubate at 37°C for 1 hour.


ComponentAmount

Expression Vector5 μg
SfiI (20 U/μL)1 μL
10× Buffer (NEB 2)2.5 μL
10× BSA2.5 μL
Nuclease-free waterto 25 μL

Total25 μL

79| Purify the digested Library or Clone insert fragment by electrophoresis on 1.5% agarose gel (1× TAE with EtBr). Cut out the appropriate band and purify by “Freeze and Squeeze”69 or with the QIAquick Gel Extraction kit.

Purify the expression vector with the PureLink PCR Purification kit (Invitrogen).

PAUSE POINT Purified vector and insert can be stored at -20°C indefinitely.

80| Ligate purified expression vector and the Library/Clone insert using T4 DNA ligase (shown below). Also set up a control ligation with no insert to assess the library background. For large libraries two to three ligation reactions may be required to reach the desired library size. Allow the ligation reaction to proceed overnight at room temperature.


ComponentLibrary LigationLibrary ControlClone LigationClone Control

Expression Vector1 μg1 μg100 ng100 ng
Library/Clone Insert750 ng-------50 ng-------
5× T4 DNA Ligase Buffer20 μL20 μL4 μL4 μL
T4 DNA Ligase (1 U/μL)10 μL10 μL1 μL1 μL
Nuclease-free waterto 100 μLto 100 μLto 20 μLto 20 μL

Total100 μL100 μL20 μL20 μL

81| For a clone ligation, follow steps 4 through 6. Next, plate 50 μL each of the transformed ligation and control onto SB plates containing 100 μg/mL carbenicillin. Allow these to grow overnight at 37°C.

Pick five colonies from the transformed ligation plates and inoculate 2.5 mL SB media containing 50 μg/mL carbenicillin. Shake (250 rpm) overnight at 37°C.

Prepare small-scale plasmid purifications (PureLink Quick Plasmid Miniprep kit from Invitrogen) from the overnight cultures and sequence with an appropriate primer.

PAUSE POINT Purified plasmid DNA can be stored at -20°C indefinitely

82| For a library ligation, ethanol precipitate the ligation and control reactions. Add 10 μL of 3 M sodium acetate (NaOAc, pH ~5.2-6.0), 1 μL glycogen (1 mg/mL) (optional), and 250 μL of absolute ethanol. Let stand at -20°C for at least 1 hour. Centrifuge at ≥ 15,000 xg for 30 minutes. Remove supernatant and wash with 1 mL 70% ethanol. Centrifuge at ≥ 15,000 xg for 15 minutes. Remove supernatant and allow pellet to air dry for 15 minutes. Resuspend DNA pellet in 10 μL nuclease-free water.

PAUSE POINT Ligations can be stored in the NaOAc/ethanol solution or after resuspension in water at -20°C indefinitely.

83| Transform entire 10-μL ligation reaction (5 μL + 100 μL of competent cells × 2 cuvettes) and 5 μL of the control in 100 μL competent cells via electroporation (2.5 kV, 15.0 μF, 200 Ohms). We recommend SS32068 cells due to their high competency.

84| After electroporation, add 1 mL SOC media each to the transformed ligations and control and transfer to 14-mL culture tubes, pooling the two transformed ligation cuvettes. Incubate with agitation (250 rpm) at 37°C for 1 hour.

85| Make a 1:100 dilution of the transformed ligation culture in SOC media. Plate 0.2 μL and 2 μL of the diluted transformed ligation culture and the transformed control culture onto SB plates containing 100 μg/mL carbenicillin. Allow these to grow overnight at 37°C. Add the remaining transformed ligation culture to 150 mL of SB with 50 μg/mL carbenicillin. Incubate the culture with agitation (250 rpm) overnight at 37°C.

86| Count colonies on the transformed ligation and control plates to ensure 10× coverage of the library. The transformed ligation plates should have at least 20-fold more colonies than the control plates to ensure less than 10% background.

?TROUBLESHOOTING

87| Repeat steps 9 and 10. The fragment released from the library should be 600 bp.

?TROUBLESHOOTING

TROUBLESHOOTING


StepsProblemsPossible ReasonSolution

9, 16, 25, 34, 43, 49, 64, 72, 77, 87Band after SfiI digestions is too large or there is more tdan one band.Multiple insertsA) Repeat ligation witd less insert;or B) Digest library witd SpeI, clean vector by gel extraction, and religate;or C) use AhdI instead of SpeI to cut tde vector and insert; tdis will give a larger insert size (see Fig. 2 for approximate AhdI location).

8, 15, 24, 33, 42, 48, 50, 54, 86Library size is too smallLow cell competencyA) Use a strain with higher competency potential;or B) make a fresh batch of competent cells;or C) do two (or more) simultaneous ligation reactions.

8, 15, 24, 33, 42, 48, 50, 54, 86Background is too highInsufficient digestion of vectorRe-digest vector with higher concentration of enzyme or for a longer period of time.

8, 15, 24, 33, 42, 48, 50, 54, 86Background is too highInsufficient dephosphorylation of vectorClean vector by running on a 1.5% agarose gel and clean via “Freeze and Squeeze”69 or electroelution.

Timing

  • Construction of a six-finger GNN library: Steps 1 – 34, 9 days
  • Construction of a five-finger GNH/ANN library: Steps 35 – 54, 11 days
  • Construction of a designed six-finger clone: Steps 55 – 77, 13 days
  • Transferring a zinc-finger library or protein to an expression vector: Steps 78 – 87, 3 days

Anticipated Results

In our experience, selection of zinc-fingers from libraries has proven to be a reliable method for discovering proteins that are able to bind specific genomic target sites44-53. Designed zinc-fingers, with domains designed based on a selected sequence, are not always able to bind their intended genomic target due to secondary structure, chromatin structure, or occlusion of the DNA sequence by other endogenous DNA-binding proteins. Selection for functional zinc finger proteins with activity in the native environment of the cell thus leads to more reliably active zinc-finger proteins. The library construction method presented in this protocol provides libraries that contain fewer mutations and less bias than libraries created using PCR-based methods or by mixing of individual zinc-fingers. These libraries can also be used in bacterial55,56, yeast57,58, and cell-free59,60 zinc-finger selection systems.

We have built a variety of libraries and specific clones than have been successfully used in different assays. Libraries constructed using this protocol have a very low background of nonfunctional proteins and insert-free vector DNA. As shown in Fig. 3, all of the randomly-selected clones analyzed in the process of building the 5ZF GNH/ANN libraries contained the correct size insert for a five-finger protein. For these libraries, the background of insert-free vector was between 1 and 5%.

Figure 3
Agarose gel electrophoresis (1.5% in 1×TAE with EtBr) of individual clones from the 5ZF GNH/ANN library. Colonies were selected randomly and tested by SfiI enzyme digestion. All clones released a single 500-bp insert after digestion, reflecting ...

To demonstrate the success of library construction, the 4ZF and 5ZF GNH/ANN libraries assembled using the described protocol and cloned into pMX-VP64-IRES-GFP and pMX-KRAB-IRES-GFP were used to retrovirally transduce MDA-MB-231 cells. A western blot of the cell extract with detection via the HA tag showed strong expression of all four libraries (Fig. 4). Toxicity problems during the construction of the libraries have been overcome by performing all the cloning in a non-expressing vector (pSCV), thus ensuring a non-biased library.

Figure 4
Western blot of 4ZF and 5ZF GNH/ANN library expression with detection via an HA tag with anti-HA-peroxidase antibody (Roche). Libraries were cloned into the pMX-IRES-GFP retroviral vectors as both VP64 and KRAB and transduced into MDA-MB-231 cells.

Supplementary Material

Supplemental Figure 1

Supplementary Figure 1:

Vector maps of SuperZiF vectors and pSCV.

Supplemental Figure 2

Supplementary Figure 2:

Vector maps of expression vectors in Table 1.

Supplemental Sequence Archive

Acknowledgments

We thank S. Juraja and S. Alonso for critical reading of the manuscript and members of our group for helpful suggestions. L.J.S. is supported by The American Cancer Society Illinois Division - Linda M. Campbell Postdoctoral Fellowship. Funding was provided by grants from the U.S. National Institutes of Health.

Footnotes

Author Contibutions B.G. and L.J.S. contributed equally to this work. C.F.B. conceived of the SuperZif library construction concept and directed the research. Y.Y., B.G. and C.F.B. designed the SuperZif vectors. R.P.F designed and constructed the pSCV vector, modified the SuperZiFCNN vector and aided in library construction. B.G., L.J.S., and L.A. constructed and tested the libraries. The paper was written by L.J.S. with assistance from B.G., C.F.B. and R.F.

Competing Interests Statement The authors declare that they have no competing financial interests.

References

1. Elrod-Erickson M, Rould MA, Nekludova L, Pabo CO. Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. Structure. 1996;4:1171–1180. [PubMed]
2. Pavletich NP, Pabo CO. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science. 1991;252:809–817. [PubMed]
3. Segal DJ, Dreier B, Beerli RR, Barbas CF., 3rd 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;96:2758–2763. [PubMed]
4. Dreier B, Beerli RR, Segal DJ, Flippin JD, Barbas CF., 3rd 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;276:29466–29478. [PubMed]
5. Dreier B, et al. Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem. 2005;280:35588–35597. [PubMed]
6. Jamieson AC, et al. Controlling gene expression in Drosophila using engineered zinc finger protein transcription factors. Biochem Biophys Res Commun. 2006;348:873–879. [PubMed]
7. Snowden AW, et al. Repression of vascular endothelial growth factor A in glioblastoma cells using engineered zinc finger transcription factors. Cancer Res. 2003;63:8968–8976. [PubMed]
8. Tan S, et al. Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity. Proc Natl Acad Sci U S A. 2003;100:11997–12002. [PubMed]
9. Bartsevich VV, Miller JC, Case CC, Pabo CO. Engineered zinc finger proteins for controlling stem cell fate. Stem Cells. 2003;21:632–637. [PubMed]
10. Beerli RR, Segal DJ, Dreier B, CF Barbas., 3rd Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc Natl Acad Sci U S A. 1998;95:14628–14633. [PubMed]
11. Beerli RR, Dreier B, Barbas CF., 3rd Positive and negative regulation of endogenous genes by designed transcription factors. Proc Natl Acad Sci U S A. 2000;97:1495–1500. [PubMed]
12. Falke D, Fisher M, Ye D, Juliano RL. Design of artificial transcription factors to selectively regulate the pro-apoptotic bax gene. Nucleic Acids Res. 2003;31:e10. [PMC free article] [PubMed]
13. Falke D, Fisher MH, Juliano RL. Selective transcription of p53 target genes by zinc finger-p53 DNA binding domain chimeras. Biochim Biophys Acta. 2004;1681:15–27. [PubMed]
14. Xu D, Ye D, Fisher M, Juliano RL. Selective inhibition of P-glycoprotein expression in multidrug-resistant tumor cells by a designed transcriptional regulator. J Pharmacol Exp Ther. 2002;302:963–971. [PubMed]
15. Rebar EJ, et al. Induction of angiogenesis in a mouse model using engineered transcription factors. Nat Med. 2002;8:1427–1432. [PubMed]
16. Liu PQ, et al. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J Biol Chem. 2001;276:11323–11334. [PubMed]
17. Gommans WM, et al. Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein-2 promoter as a novel anti-cancer treatment. Mol Carcinog. 2007;46:391–401. [PubMed]
18. Nomura W, Barbas CF., 3rd In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase. J Am Chem Soc. 2007;129:8676–8677. [PubMed]
19. Smith AE, Ford KG. Specific targeting of cytosine methylation to DNA sequences in vivo. Nucleic Acids Res. 2007;35:740–754. [PMC free article] [PubMed]
20. Minczuk M, Papworth MA, Kolasinska P, Murphy MP, Klug A. Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase. Proc Natl Acad Sci U S A. 2006;103:19689–19694. [PubMed]
21. Snowden AW, Gregory PD, Case CC, Pabo CO. Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo. Curr Biol. 2002;12:2159–2166. [PubMed]
22. Gordley RM, Smith JD, Graslund T, Barbas CF., 3rd Evolution of programmable zinc finger-recombinases with activity in human cells. J Mol Biol. 2007;367:802–813. [PubMed]
23. Akopian A, He J, Boocock MR, Stark WM. Chimeric recombinases with designed DNA sequence recognition. Proc Natl Acad Sci U S A. 2003;100:8688–8691. [PubMed]
24. Yant SR, Huang Y, Akache B, Kay MA. Site-directed transposon integration in human cells. Nucleic Acids Res. 2007;35:e50. [PMC free article] [PubMed]
25. Tan W, Dong Z, Wilkinson TA, Barbas CF, 3rd, Chow SA. Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells. J Virol. 2006;80:1939–1948. [PMC free article] [PubMed]
26. Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A. 1996;93:1156–1160. [PubMed]
27. Bibikova M, et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol. 2001;21:289–297. [PMC free article] [PubMed]
28. Bibikova M, Golic M, Golic KG, Carroll D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics. 2002;161:1169–1175. [PubMed]
29. Bibikova M, Beumer K, Trautman JK, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science. 2003;300:764. [PubMed]
30. Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science. 2003;300:763. [PubMed]
31. Porteus MH. Mammalian gene targeting with designed zinc finger nucleases. Mol Ther. 2006;13:438–446. [PubMed]
32. Lloyd A, Plaisier CL, Carroll D, Drews GN. Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc Natl Acad Sci U S A. 2005;102:2232–2237. [PubMed]
33. Wright DA, et al. High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J. 2005;44:693–705. [PubMed]
34. Urnov FD, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005;435:646–651. [PubMed]
35. Alwin S, et al. Custom zinc-finger nucleases for use in human cells. Mol Ther. 2005;12:610–617. [PubMed]
36. Moehle EA, et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci U S A. 2007;104:3055–3060. [PubMed]
37. Beerli RR, Schopfer U, Dreier B, Barbas CF., 3rd Chemically regulated zinc finger transcription factors. J Biol Chem. 2000;275:32617–32627. [PubMed]
38. Ghosh I, Stains CI, Ooi AT, Segal DJ. Direct detection of double-stranded DNA: Molecular methods and applications for DNA diagnostics. Mol Biosyst. 2006;2:551–560. [PubMed]
39. Ooi AT, Stains CI, Ghosh I, Segal DJ. Sequence-enabled reassembly of beta-lactamase (SEER-LAC): a sensitive method for the detection of double-stranded DNA. Biochemistry. 2006;45:3620–3625. [PMC free article] [PubMed]
40. Stains CI, Furman JL, Segal DJ, Ghosh I. Site-specific detection of DNA methylation utilizing mCpG-SEER. J Am Chem Soc. 2006;128:9761–9765. [PubMed]
41. Liu Q, Segal DJ, Ghiara JB, Barbas CF., 3rd Design of polydactyl zinc-finger proteins for unique addressing within complex genomes. Proc Natl Acad Sci U S A. 1997;94:5525–5530. [PubMed]
42. Guan X, et al. Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors. Proc Natl Acad Sci U S A. 2002;99:13296–13301. [PubMed]
43. Segal DJ, Crotty JW, Bhakta MS, Barbas CF, 3rd, Horton NC. Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA. J Mol Biol. 2006;363:405–421. [PubMed]
44. Blancafort P, Magnenat L, Barbas CF., 3rd Scanning the human genome with combinatorial transcription factor libraries. Nat Biotechnol. 2003;21:269–274. [PubMed]
45. Magnenat L, Blancafort P, Barbas CF., 3rd In vivo selection of combinatorial libraries and designed affinity maturation of polydactyl zinc finger transcription factors for ICAM-1 provides new insights into gene regulation. J Mol Biol. 2004;341:635–649. [PubMed]
46. Lee DK, Kim YH, Kim JS, Seol W. Induction and characterization of taxol-resistance phenotypes with a transiently expressed artificial transcriptional activator library. Nucleic Acids Res. 2004;32:e116. [PMC free article] [PubMed]
47. Zhao XH, Zhu XD, Liu J, Rao XJ. & Huang, P.T. [Construction of a SV40 promoter specific artificial transcription factor] Sheng Wu Gong Cheng Xue Bao. 2003;19:608–612. [PubMed]
48. Blancafort P, et al. Genetic reprogramming of tumor cells by zinc finger transcription factors. Proc Natl Acad Sci U S A. 2005;102:11716–11721. [PubMed]
49. Lund CV, Blancafort P, Popkov M, Barbas CF., 3rd Promoter-targeted phage display selections with preassembled synthetic zinc finger libraries for endogenous gene regulation. J Mol Biol. 2004;340:599–613. [PubMed]
50. Lindhout BI, Pinas JE, Hooykaas PJ, van der Zaal BJ. Employing libraries of zinc finger artificial transcription factors to screen for homologous recombination mutants in Arabidopsis. Plant J. 2006;48:475–483. [PubMed]
51. Park KS, et al. Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors. Nat Biotechnol. 2003;21:1208–1214. [PubMed]
52. Park KS, Jang YS, Lee H, Kim JS. Phenotypic alteration and target gene identification using combinatorial libraries of zinc finger proteins in prokaryotic cells. J Bacteriol. 2005;187:5496–5499. [PMC free article] [PubMed]
53. Blancafort P, et al. Modulation of drug resistance by artificial transcription factors. Mol Cancer Ther. 2008;7:688–697. [PubMed]
54. Tschulena U, Peterson KR, Gonzalez B, Fedosyuk H, Barbas CF., III Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production. Nature Structure and Molecular Biology. 2009;16:1195–1199. [PMC free article] [PubMed]
55. Meng X, Thibodeau-Beganny S, Jiang T, Joung JK, Wolfe SA. Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method. Nucleic Acids Res. 2007;35:e81. [PMC free article] [PubMed]
56. Durai S, Bosley A, Abulencia AB, Chandrasegaran S, Ostermeier M. A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions. Comb Chem High Throughput Screen. 2006;9:301–311. [PubMed]
57. Bae KH, Kim JS. One-step selection of artificial transcription factors using an in vivo screening system. Mol Cells. 2006;21:376–380. [PubMed]
58. Bartsevich VV, Juliano RL. Regulation of the MDR1 gene by transcriptional repressors selected using peptide combinatorial libraries. Mol Pharmacol. 2000;58:1–10. [PubMed]
59. Ihara H, et al. In vitro selection of zinc finger DNA-binding proteins through ribosome display. Biochem Biophys Res Commun. 2006;345:1149–1154. [PubMed]
60. Sepp A, Choo Y. Cell-free selection of zinc finger DNA-binding proteins using in vitro compartmentalization. J Mol Biol. 2005;354:212–219. [PubMed]
61. Carroll D, Morton JJ, Beumer KJ, Segal DJ. Design, construction and in vitro testing of zinc finger nucleases. Nat Protoc. 2006;1:1329–1341. [PubMed]
62. Wright DA, et al. Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protoc. 2006;1:1637–1652. [PubMed]
63. Maeder ML, et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell. 2008;31:294–301. [PMC free article] [PubMed]
64. Desjarlais JR, Berg JM. Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins. Proc Natl Acad Sci U S A. 1993;90:2256–2260. [PubMed]
65. Mandell JG, Barbas CF., 3rd Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 2006;34:W516–523. [PMC free article] [PubMed]
66. Gordley RM, Gersbach CA, Barbas CF., 3rd Synthesis of programmable integrases. Proc Natl Acad Sci U S A. 2009;106:5053–5058. [PubMed]
67. Liu X, et al. Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc Natl Acad Sci U S A. 1997;94:10669–10674. [PubMed]
68. Sidhu SS, Lowman HB, Cunningham BC, Wells JA. Phage display for selection of novel binding peptides. Methods Enzymol. 2000;328:333–363. [PubMed]
69. Barbas CF, 3rd, Burton DR, Scott JK, Silverman GJ. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press; 2001.