Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies

Abstract

The understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein-DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular assembly method to engineer artificial proteins and enzymes that activate, repress, or create defined changes to user-specified genes in human cells, plants, and other organisms. We have also engineered novel methods for externally controlling protein activity and delivery, as well as developed new strategies for the directed evolution of protein and enzyme function. This Account summarizes our work in these areas and highlights independent studies that have successfully used the modular assembly approach to create proteins with novel function. We also discuss emerging alternative methods for genomic targeting, including transcription activator-like effectors (TALEs) and CRISPR/Cas systems, and how they complement the synthetic zinc finger protein technology.

Figure 1

Structure and applications of zinc finger proteins. (Left) The designed six-finger zinc finger protein, Aart (light brown), in complex with target DNA (gray) (PDB ID: 2I13). The inset shows a single zinc finger domain. The side-chains of the conserved Cys and His residues that coordinate with a Zn ion (red sphere) are shown as sticks. (Right) Cartoon illustrating the applications of zinc finger technology.

Figure 2

Phage-display selection of zinc finger proteins. Highly diverse three-finger zinc finger libraries were generated by randomization of the α-helical residues (−1, 1, 2, 3, 5, and 6) of the central zinc finger. These zinc finger libraries were then displayed on the surface of phage and incubated with biotinylated hairpin DNA targets. Phage-display libraries were subjected to stringent selection pressure to ensure sequence specificity. Phages that bound to single biotinylated DNA targets were recovered and amplified, and the selection process was repeated.

Figure 3

Summary of the selected zinc finger domains used for modular assembly. The α-helical residues (−1, 1, 2, 3, 5, and 6) for each zinc finger are shown. Positions −1, 3, and 6 are underlined.

Figure 4

Specificity profiles of the zinc finger domains selected or designed to recognize each of the 16 possible 5′-GNN-3′ triplets. Blue bars represent binding to all 16 possible 5′-GNN-3′ triplets. Red bars represent binding to pools of 5′-GNN-3′, 5′-ANN-3′, 5′-CNN-3′, and 5′-TNN-3′ triplets. Data previously published in refs (5) and (12).

Figure 5

Contacts between the recognition helices of Aart, a designed six-finger zinc finger protein, and target DNA. The α-helical residues that specifically interact with DNA are shown as purple sticks. All residues are numbered according to their α-helical position (−1, 3, or 6). DNA is shown as orange and yellow sticks. The indicated DNA triplet and the α-helical residues specific for that target are indicated above each structure.

Figure 6

Zinc finger nuclease (ZFN) structure. (A) (Top) Three-dimensional model of the ZFN dimer (purple and blue) in complex with DNA (gray) (PDB IDs: 1FOK and 2I13, respectively). (Bottom) Cartoon of the ZFN dimer bound to DNA. (B) Model of the FokI cleavage domain dimer (purple and blue) in complex with DNA (PDB ID: 2FOK). Sharkey mutations (S418P and K441E) are shown as yellow spheres. The catalytic amino acids Asp 450, Asp 467, and Lys 469 are shown as red sticks.

Figure 7

Zinc finger recombinase (ZFR) structure. (A) (Top) Three-dimensional model of the ZFR dimer (blue and orange) in complex with DNA (gray), adapted from Gaj et al. (PDB IDs: 1GDT and 2I13, respectively). (Bottom) Cartoon of the ZFR dimer bound to DNA. (B) Residues that confer recombinase catalytic specificity and subject to reprogramming for recognition of new sequences are shown as spheres. Carbon, oxygen and nitrogen atoms are colored orange, red and blue, respectively. DNA is shown as gray sticks.