The generation of zinc finger proteins by modular assembly

Abstract

The modular assembly (MA) method of generating engineered zinc finger proteins (ZFPs) was the first practical method for creating custom DNA-binding proteins. As such, MA has enabled a vast exploration of sequence-specific methods and reagents, ushering in the modern era of zinc finger-based applications that are described in this volume. The first zinc finger nuclease to cleave an endogenous site was created using MA, as was the first artificial transcription factor to enter phase II clinical trials. In recent years, other excellent methods have been developed that improved the affinity and specificity of the engineered ZFPs. However, MA is still used widely for many applications. This chapter will describe methods and give guidance for the creation of ZFPs using MA. Such ZFPs might be useful as starting materials to perform other methods described in this volume. Here, we also describe a single-strand annealing recombination assay for the initial testing of zinc finger nucleases.

Fig. 1

Construction of ZFNs by MA. (A) A schematic diagram of the hydrogen bonding between the ZFP GZF3 and its 9-bp DNA target site. The ZFP binds “anti-parallel” (C-term. to N-term.) with respect to the DNA strand containing the target sequence (5’ to 3’). Vertical lines are presumed hydrogen bonds. The diagonal line represents a Target Site Overlap (TSO) by Finger 3, influencing the 5’ base of the neighboring Finger-2 subsite to be G. Fingers with potential TSO interactions are indicated in Tables 1–4. (B) A heterodimeric ZFN. Each monomer binds in an everted orientation so that the FokI cleavage domains at their C-termini (open ovals) can dimerize and cleave the DNA within the 6-bp spacer (TTTAAA) between the ZFP sites. (C) The sequence of the three-finger ZFP GZF1, created by inserting the Barbas modules (in brackets) into the Sp1C scaffold. Regions corresponding to beta strands (arrows), alpha helix (tube), and the interfinger linker (TGEKP, wavy line) are indicated. (D) The sequence for GZF1 using the Sangamo modules inserted into the Zif268 scaffold. (E) The sequence of GZF1 using the ToolGen modules, which consist of full-length fingers (in brackets) joined by interfinger linkers. Noted that D and E are for illustrative purposes; they have not been evaluated for function. (F) Additional protein sequences must be added to the ZFPs in panels C, D, or E, and must be encoded by the specific DNA sequences indicated in order to allow proper cloning into the ZFP cloning vector. (G) A schematic diagram of the ZFP cloning vector. A PGK promoter (PGK pro), HA tag (HA), SV40 Nuclear Localization Signal (NLS), ZFP (GZF1), and FokI cleavage domain (here shown to contain the obligate heterodimer modification RR), are indicated. PCR primers used for sequencing are shown in gray.

Fig. 2

The SSA assay. (A) A schematic diagram of the SSA reporter plasmid. The sequence at the junction site between the homologous repeats is shown at the top. The GZF1 and GZF3 heterodimer target site is indicated. PCR primers used in construction are shown as black arrows on the plasmid diagram below. PCR primers used for amplification of the junction region and sequencing are shown in gray. (B) Schamatics of the four plasmids that will be transfected into the test cells for the SSA assay. (C) The expressed ZFN pair will bind at the target site and create a double strand break (DSB). The break will be efficiently repaired by the SSA pathway to reconstruct an active firefly luciferase reporter gene.

Fig. 3

Exemplary data from an SSA assay. Firefly (SSA recombination due to ZFN activity) and Renilla (constitutively high in the absence of ZFN toxicity) luciferase data is shown for some of the experiments described in Note 1, including the analysis of GZF1-wt and GZF3-wt homodimers, as well as GZF1-wt/GZF3-wt and GZF1-RR/GZF3-DD heterodimers. Luciferase activity was measured 24 h post-transfection. Error bars indicate the standard deviation of triplicate experiments.