- Genome editing with engineered zinc finger nucleases.
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- Genome Editing with engineered nucleases
- Stimulating Gene Targeting With Double-Strand Breaks
- Genome Engineering With Zinc-Finger Nucleases
- Zinc finger nuclease
ZFN-induced double-strand breaks are subject to cellular DNA repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies.
This article briefly reviews the history of ZFN development and summarizes applications that have been made to genome editing in many different organisms and situations.
Considerable progress has been made in methods for deriving zinc-finger sets for new genomic targets, but approaches to design and selection are still being perfected. An issue that needs more attention is the extent to which available mechanisms of double-strand break repair limit the scope and utility of ZFN-initiated events.
The bright prospects for future applications of ZFNs, including human gene therapy, are discussed. Genetics is driven by the ability to connect genotype with phenotype. The classical approach is to identify a novel phenotype, whether occurring spontaneously or derived by mutagenesis, to identify the responsible gene s and to discover why mutations at that locus have the observed effect. A more modern approach, sometimes called reverse genetics, is to identify a gene from a genomic sequence to make mutations specifically in that gene and to characterize the resulting phenotype.
Two types of gene-specific manipulations can be envisioned Figure 1.
In targeted gene replacement, the goal is to replace an existing sequence with one designed in the laboratory. The latter allows the introduction of both more subtle and more extensive alterations.
Genome editing with engineered zinc finger nucleases.
Illustration of two types of genome engineering. In the top portion, the horizontal line represents a genome segment, and the open rectangles, two individual genes. The jagged arrow on the left indicates an unspecified mutagenic agent targeted to one gene.
The shaded rectangle on the right is a manipulated version of the second gene that has been supplied by the experimenter.
Success in this case depends on several features: the ability to manipulate segments of yeast DNA in the laboratory, the ability to introduce DNA into yeast cells, interaction between donor and target DNA by homologous recombination, the near absence of competing reactions that would integrate the donor into alternative sites in the genome, and the ability to apply strong selection for the desired product.
These properties are shared by some other fungi and many bacteria, but not by the majority of eukaryotic organisms. Making targeted gene replacements has also become standard practice in mice, thanks to the availability of embryonic stem ES cells that can be manipulated in culture and the development of powerful selection procedures Capecchi Like targeting in yeast, the process in mice depends on homologous recombination between the donor and the target.
In addition, selection must be applied against the more common products of random integration.
This is accomplished by placing a positive selectable marker inside the donor homology and a negative selectable marker outside the homology Mansour et al. Double selection yields the desired replacements, and the pluripotency of the ES cells allows them to populate all cell lineages after injection into early embryos.
In both yeast and mouse cells, the absolute frequency of homologous recombination between donor and target sequences is quite low—on the order of one in every 10 4 to 10 7 cells.
Selection in culture allows the recovery of the rare cells that have enjoyed the desired event. With other experimental organisms, ES cells are not available, screening or selection procedures are not adequate, and development of useful gene-targeting approaches is impeded by the low frequency of recombination.
The challenge in extending gene targeting to other organisms and situations could be viewed largely as one of increasing the frequency of recombination. This could be done a priori by manipulating the donor DNA, the genomic target, or the genetic background.
This makes sense, as DNA ends are typically recombinagenic, but the effect is rather modest. Increasing the amount of donor DNA has little effect, and in mammalian cells seems largely to increase the frequency of nonhomologous integration Vasquez et al.
The use of oligonucleotide donors to introduce very localized changes has been somewhat successful, but the high frequencies claimed in early reports have not proved robust or reproducible.
Some attempts have been made to increase the levels of proteins involved in recombination reactions, again with limited success. The greatest impediment to efficient targeting is the fact that an intact target is essentially inert.
This has been demonstrated by damaging the target and observing increased levels of recombination. Early experiments showed that DNA-damaging agents stimulated homologous exchanges between sister chromatids Latt Most compelling, however, were studies showing that a single double-strand break DSB dramatically increased the frequency of local recombination.
These experiments were inspired by the discovery that natural recombination events, including meiotic crossing over and mating-type switching in yeast, are initiated by DSBs. In this approach, pioneered by Haber in yeast Rudin et al. Recombination with a homologous donor DNA was stimulated by several orders of magnitude.
Other means of damaging the target have also shown some utility, but nothing as effective as making a DSB.
Chromosomal breaks are detected in cells as potentially lethal damage, and one natural pathway of DSB repair is copying from a homologous template.
From this perspective, DSB-stimulated gene targeting simply provides an exogenous template for a natural repair process. An alternative repair pathway for DSBs, nonhomologous end joining, often joins the broken ends inaccurately, creating deletions, insertions, and substitutions at the break site.
Thus, both mutagenesis and gene replacement are stimulated locally by DSBs Figure 2. Repair outcomes of a genomic double-strand break, illustrated for the case of ZFN cleavage. A pair of three-finger ZFNs is shown at the top in association with a target gene open box. If a homologous donor DNA is provided solid box, left , repair can proceed by homologous recombination using the donor as template.
The amount of donor sequence ultimately incorporated will typically decline with distance from the original break, as illustrated by the shading. Alternatively, the break can be repaired by nonhomologous end joining, leading to mutations at the cleavage site.
These may be deletions, insertions, and base substitutions, usually quite localized, but sometimes extending away from the break. While they provided very useful information on the efficiency and mechanisms of DSB repair, they were limited in their utility because their recognition sites had to be inserted in the genome by a low-efficiency process before they could be used to effect high-efficiency recombination.
Reagents were needed that could be designed to attack arbitrarily chosen, preexisting genomic sequences.
A number of research groups focused on small compounds that would find their targets essentially by base recognition. These included oligonucleotides that could form DNA triplexes by adding a synthetic strand to a duplex target Chin and Glazer Variations on the theme included peptide nucleic acids that substitute a peptide backbone for the usual sugar—phosphate linkage Kim et al.
These recognition moieties were linked to reactive groups that would cut or locally damage the DNA, thereby stimulating repair by homologous recombination. These efforts have yielded some success, but they are limited in the number of target sequences that they can access, and the frequencies of site-specific damage have not been consistently high.
Another approach has been to modify the recognition specificity of enzymes such as I- Sce I homing endonucleases, also called meganucleases Ashworth et al. This has proved very successful in some cases, but the intimate connection between the recognition and cleavage elements in the protein structures makes it challenging to alter one without affecting the other.
These synthetic proteins originated in the observation by Chandrasegaran that the natural type IIS restriction enzyme, Fok I, has physically separable binding and cleavage activities Li et al. The cleavage domain has no apparent sequence specificity, and Chandrasegaran showed that cutting could be redirected by substituting alternative recognition domains for the natural one Kim and Chandrasegaran ; Kim et al.
The crystal structure of a set of three fingers bound to DNA showed that each finger contacts primarily 3 bp of DNA in remarkably modular fashion Pavletich and Pabo This suggested that many different sequences could be attacked by making novel assemblies of ZFs.
Zinc fingers are shown as open boxes, with short vertical lines indicating the main contacts with the DNA base pairs. Fok I cleavage domains are shown as shaded boxes, with common cleavage sites, spaced by 4 bp, and indicated by vertical arrows. Zinc fingers are numbered from the N terminus.
The linker between the binding and cleavage domains of one protein is labeled. The spacer between the zinc-finger binding sites, 6 bp in this case, is also indicated. Each zinc finger is shown in a shade of pink, in ribbon representation on the left and space-filling representation on the right.
The Fok I cleavage domains are shown in shades of blue. The four-amino-acid linker between the binding and cleavage domains is gray. DNA is shown with the sugar—phosphate backbone in orange and the bases in orange and blue.
Genome editing with engineered zinc finger nucleases pdf to jpg
The separation between ZF binding sites is 6 bp. This model Smith et al. I am grateful to Dr.
Genome Editing with engineered nucleases
Frank Whitby for help with the modeling. Although it was not recognized initially Kim et al. The dimer interface is weak, and the best way to achieve cleavage is to construct two sets of fingers directed to neighboring sequences and join each to a monomeric cleavage domain Figure 3.
When both sets of fingers bind to their recognition sequences, high local concentration facilitates dimerization and cleavage. Several studies have shown that the optimum configuration uses a short linker between the domains of the protein and a spacer of 5 or 6 bp 7 can also work between binding sites that lie in inverted orientation Bibikova et al.
Stimulating Gene Targeting With Double-Strand Breaks
The requirement for dimerization is a great advantage for this reason: because a monomer is not active, cleavage does not occur at single binding sites. The cleavage reagent is assembled only at the target if the fingers have adequate specificity, and the combined requirement for binding two proteins brings the overall specificity into a very useful range; e. The first ZFNs were created as chimeric restriction endonucleases and were shown to have in vitro activity Kim et al.
It was not clear that the prokaryotic cleavage domain would be able to act on DNA assembled into chromatin, but experiments in Xenopus oocytes with a synthetic, extrachromosomal substrate and ZFNs of known specificity showed very high efficiency of cleavage and recombination Bibikova et al.
The first success with a ZFN pair designed de novo for a genomic target occurred in Drosophila.
Genome Engineering With Zinc-Finger Nucleases
Both targeted mutagenesis Bibikova et al. Since then, ZFN pairs have been designed, constructed, and used successfully for individual genes in quite a variety of organisms and cell types Table 1. TM refers to targeted mutagenesis by nonhomologous end joining TGR is targeted gene replacement by homologous recombination. In addition to the examples shown here, I have heard reliable, but unpublished, reports of successful ZFN-induced targeting in several other organisms.
The list of references is not exhaustive, but provides guidance to key publications. In cultured cells, expression constructs for ZFNs use promoters appropriate to the cell type and vectors that can be introduced by transfection of DNA or infection by viruses. The same methods also serve to introduce the donor. This required rather elaborate strain construction, and a welcome breakthrough occurred when it was demonstrated that excellent efficiencies of both homologous and nonhomologous events could be obtained by injecting ZFN mRNAs and donor DNA into embryos Beumer et al.
This is a well-established method in zebrafish, and very usable frequencies of ZFN-induced mutagenesis have been achieved in quite a number of genes Doyon et al.
Recent experiments with embryos of rat Geurts et al.
Zinc finger nuclease
Injection into silkworm embryos, very much in parallel with the Drosophila method, also works Takasu et al. Homologous recombination with donor DNA has been achieved in rats and mice Meyer et al.
In all these cases, viable adults carrying germline mutations were grown from the treated embryos. In other organisms, more specialized approaches to delivery have been taken. In plants—both the favored experimental cress, Arabidopsis thaliana , and some crop species—ZFN expression was achieved by delivering coding sequences under the control of a viral promoter by agrobacterial transformation Lloyd et al.