Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells

Abstract

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting--the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for 'directed mutagenesis' and targeted 'gene editing' of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.

Figure 1

Structures of an individual ZF motif (A) and a four-finger ZFP, which is formed by linking four such individual ZF motifs (B). Zif268 bound to its cognate DNA site (C) and potential binding of Zif268 to degenerate sites (D). The key base contacts were deduced from the crystal structure of Zif268–DNA complex (44). Each finger makes contact with its target 3 bp site. In addition, Asp² at position 2 in each finger makes contact with a base outside the 3 bp site (C). Fingers 1 and 3 of Zif268 make specific contacts only with two bases of their cognate DNA triplets, while specific base contacts are seen with all the three bases of finger 2. Zif268 could potentially bind to other secondary or degenerate sites (D) as indicated, where N = G, A, T or C.

Figure 2

ZFP platform technology. Other functionalities like non-specific FokI cleavage domain (N), transcription activator domain (A), transcription repressor domain (R) and methylases (M) can be fused to ZFPs to form respectively ZFN, ZFA, ZFR and ZFM. The literature references for each of these chimeric ZFPs and their potential biological applications are given in brackets.

Figure 3

Strategies for the production of ZFPs with desired DNA-binding specificity [adapted from reference (14)]. (A) Parallel selection; (B) Sequential selection; and (C) Bipartite selection. Parallel selection (A) is based on functional independence of individual ZF motifs in the ZFPs, i.e. each individual finger in a ZFP binds to its recognition site independent of its neighboring finger. In ‘sequential’ selection strategy (B), individual fingers are selected in the context of its neighbors, and thereby, circumvent the constraints placed by the target site overlap problem (Figure 1). However, this requires construction of multiple ZFP libraries and multiple selections for each and every ZFP that is desired, which is a tedious and time-consuming effort. The ‘bipartite’ strategy (C) utilizes two pre-generated ZFP libraries in each of which one-and-a half fingers of a three-finger ZFP are partially randomized at the key residues that make contact with DNA. The N-terminal part of the ZFP is randomized in one library while the C-terminal half is randomized in the other. Selection is done in parallel using the 5′ and 3′ halves, respectively of the target sequence. Selections from the individual libraries are then recombined and selected again using the full target site to obtain the ZFP with the desired specificity and affinity. Although they yield the desired high affinity ZFPs, all these selection approaches are very labor intensive and too cumbersome to perform routinely and require special expertise. Asterisks indicate pre-selected libraries.

Figure 4

One-hybrid system for detection of ZF–DNA interactions (A) Schematic of genetic selection system for interrogating ZF–DNA interactions. (B) Plasmids for one-hybrid genetic selection system. The reporter gene, either chloramphenicol acetyltransferase (CAT), or GFP is located downstream from a weak lac derivative promoter (P_wk) on pDB series plasmids. A 9 bp target site for binding by the ZF is located at a specific distance from the start of transcription. On the pA series of plasmids, the gene for the ZF is fused to a fragment of the α-subunit of RNA polymerase (rpoA[1–248]) via a sequence coding for an amino acid linker. Binding of the RpoA[1–248]–ZF fusion to the 9 bp site in the reporter plasmid recruits the other RNA polymerase subunits to stimulate transcription of the reporter gene.

Figure 5

A three-finger ZFN (called ΔQNK–FN) bound to its target sequence. Since the three-finger ZFN requires two copies of the 9 bp recognition sites in an inverted orientation in order to dimerize and produce a DSB, it effectively has an 18 bp recognition site. The three-finger ZFP (ΔQNK) was fused to the FokI cleavage domain (N or FN) to create the ZFN, ΔQNK–FN (54).

Scheme 1

Framework for DSB repair in mammalian cells.

Figure 6

ZFN-induced homology-directed DSB repair in mammalian cells. Initially, gene ‘a’ is undamaged. The repair of the gene by HR occurs after the induction of a DSB. The DSB may arise spontaneously such as by damage from reactive oxygen species during normal metabolism, induced randomly by the exposure to ionizing radiation, or induced specifically by ZFNs. The DSB is then processed to form free 3′ single-strand tails, a process that requires the Mre11/Rad50/Nbs1 complex. The HR machinery, through the actions of the strand invasion protein, Rad51, then uses the free 3′ ends to invade a homologous repair template/donor. How the machinery identifies a homologous repair donor remains unclear but it is likely that simple physical proximity plays an important role. In the normal repair of a DSB, the repair donor is the sister-chromatid and thus the template is identical to the damaged allele. In gene targeting, the repair donor would be an extra-chromosomal piece of DNA that could have sequence differences. In this figure the letter ‘b’ and dark bars denotes those sequence differences. After stand invasion, primed DNA synthesis occurs to generate new undamaged DNA using the undamaged DNA as a template. The process is completed by the annealing of the new strand of DNA with its original partner and subsequent use of that new DNA to template DNA synthesis. Through this process, original allele (‘a’) is converted into the repair allele (‘b’) while the undamaged allele is unchanged. This donation of information from the undamaged allele to the damaged allele is called gene conversion.

Figure 7

Diagram of the GFP gene targeting reporter system. ZFN-mediated gene targeting results in the conversion of GFP negative cells into GFP positive cells, which is detected using flow cytometry. GFP*, mutated GFP gene; tGFP, truncated GFP gene.

Figure 8

ZFN-mediated directed mutagenesis in human cells. (A) Targeted disruption of the CCR5 gene by NHEJ (mutagenic repair) using engineered ZFNs. Cells are transfected with ZFNs alone. CCR5 (m) depicts mutant CCR5 gene. (B) Targeted disruption of the CCR5 gene by ZFN-induced homology-directed repair. In this experiment, cells are transfected with both ZFN and CCR5Δ32 (or mutant CCR5 DNA).