Targeted Genome Editing Using Engineered Zinc Finger Nucleases

The determination of complete genome sequences for a wide variety of experimental organisms established a first step in the search for an in-depth understanding of the complex genetic functions that define living entities. Extensive genetic, biochemical, cytological and physiological analyses are now required to correlate genome sequence with the next level of understanding of genetic function. As such, technologies that allow researchers to routinely and efficiently edit the genomes of virtually any species, by directing mutations in a truly targeted fashion, would greatly enhance the understanding of basic biology, and potentially lead to novel ways of treating human disease.

Early Approaches to Targeted Genome Editing

While early approaches to genetic manipulation used random and/or non-targeted methods such as ionizing radiation and chemical-induced mutagenesis to make changes to the genome, more recent methods have employed targeted methods of genomic editing. The most well studied methods have relied on the process of homologous recombination (HR), a naturally occurring DNA-repair mechanism found in most cells that uses the second copy of a chromosome as a template to repair damage to a gene.

Starting with Nobel Prize winner Mario Capecchi’s earliest work, it was discovered that the HR process could be harnessed and directed in mouse embryonic stem cells (MESCs) by introducing exogenous DNA as a repair template to induce precise point mutations, gene deletions, and gene insertions. This approach to gene targeting led to the development of knockout mice as a research tool, still considered the gold standard for many functional genomics studies. However, HR was found to be woefully inefficient in many other relevant model systems, including mammalian somatic cells, in which the rate of HR is much lower than the rate of HR in MESCs. For this reason HR never found its footing as a robust means of mutatgenesis in cell types commonly used in functional genomics, target validation, and protein production settings.

The Zinc Finger as a DNA Targeting Tool

In the early 1990s, the discovery and manipulation of zinc finger protein domains started a field of research that enabled a targeted approach to genetic manipulation in higher level eukaryotes that was one thousand times more efficient than the prevailing HR-based mechanism. Zinc fingers are structures of about 30 amino acids held together by a zinc ion, and they determine the binding specificity of the most abundant class of transcription factors found in a wide range of species. Each zinc finger binds to a specific set of three bases.

Zinc Finger Nucleases: Highly Specific ‘Genomic Scissors’

More important was the discovery that the DNA-binding properties of zinc fingers could be fused to proteins with DNAcutting function. For instance, four zinc finger units can be assembled into a zinc finger chain that recognizes a 12 base pair DNA sequence. This chain can then be fused to the catalytic domain of the endonuclease FokI to create a zinc finger nuclease (ZFN). Because the endonuclease sub-unit of Fokl must dimerize to cleave DNA, ZFNs are designed as a pair, to bind to adjacent sequences in the target DNA with precise sequence specificity and spacing, so that cleavage occurs only at the desired target site, in essence creating a pair of specific ‘genomic scissors’ (Figure 1).

ZFN-mediated Genome Editing

The power of a ZFN-induced double-strand break (DSB) comes from the fact that a DSB can be repaired by one of two repair processes: non-homologous end joining (NHEJ) or homologydependent repair (HDR). In the absence of an available repair template, NHEJ is the preferred mechanism for DNA repair, as it produces localized mutations due to deletion and/or insertion of short sequences at the break site, some of which may result in disruption of gene function.^1-3

Alternatively, if an insertion of a transgene into the user-defined spot in the genome is desired, a donor DNA template with ends homologous to the sequences at the break site can be co-delivered into the cell along with the ZFNs, and the DSB repaired using HDR – during which the frequency of homologous recombination is >1000-fold higher than in the absence of DNA repair. This can result in as much as 20% of the cells containing a gene insertion at the target site (Figure 2).

ZFN-mediated gene targeting is a powerful and versatile tool for targeted genome editing of a wide range of organisms and cells. The process of ZFN-mediated gene modification is as simple as a single transfection experiment, and edits occur in as fast as three days. With as much as 20% of the cells undergoing targeted genomic editing, selection markers are most often unnecessary. Once high-quality ZFNs have been constructed, isogenic stable cell lines containing the modifications can be generated in as fast as one month through simple dilution cloning.

Targeted genome editing with ZFNs has a wide variety of applications, and three key modes of action have been a focus thus far: targeted gene knockouts, targeted gene integration, and targeted gene correction.

Targeted Gene Knockout

Gene knockout – the complete disruption of gene function – is the most powerful tool for determining gene function or for permanently modifying the phenotype of a cell. In addition, gene knockouts can be created in multiple cell lines in order to identify and/or validate a potential drug target, and provide a way to completely delete the functions of genes that are not amenable to technologies such as RNAi.

ZFNs offer a rapid single-step approach to targeted gene knockout in mammalian cells, using NHEJ. As an example, ZFNs have been used to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line diploid for a functional DHFR generated gene. CHO cells were chosen for this experiment as they are often the system of choice for the production of therapeutic recombinant proteins. In this particular study, gene disruption at a frequency of >1% was obtained without the use of selection markers, and studies were done to confirm that deletion of the gene at the DNA level did indeed translate into an phenotype expected of a DHFR– cell line.⁴

Additionally, ZFN-mediated gene knockout may be a robust approach across several different species, as illustrated by its successful application to Zebra fish.⁵ In another proof-of-concept experiment, ZFNs targeting the zebrafish golden and no tail/ Brachyury (ntl) genes were designed. Up to 32% of embryos injected with ZFNs targeting golden carried clones of unpigmented cells in the eye, representing loss of golden gene function. Injecting the ZFN targeting the ntl gene resulted in 27% of the embryos showing the stunted tail growth phenotype. With alternative knockout methodologies such as antisense and RNAi, possible toxicity effects of long-term expression of these polynucleotides, as well as the incomplete phenotypic penetrance that may arise from partial knockdown using these methods, may prove undesirable. In such cases, rapid and permanent gene knockout using ZFNs can offer distinct advantages.

Targeted Gene Integration

ZFN-mediated gene integration allows users to place exogenous genetic sequences precisely into targeted sites of the genome. This newfound ability will not only lead to a new generation of functional genomics studies, but will have great impact in the drug discovery world as it will the allow creation of new cell lines containing fusion tags or reporters that can be used for more efficient cell-based screening.

To gain acceptance as a robust methodology, any means of targeted gene integration should occur at a frequency high enough to obviate the need for time-consuming selection of cells carrying the desired gene. Indeed, using the ZFN methodology, targeted gene integration frequencies of 15, 6 and 5% have been observed, respectively, for insertion of a 12 bp tag, a 900 bp open reading frame and a 1.5 kb promoter-transcription unit at a specific location in human cells, without selection for desired recombinants.Targeted Gene Integration.⁷ In addition to holding promise for gene therapy research, this proof of principle makes ZFN-mediated gene integration available as a tool to researchers who need to insert genetic material into a wide variety of organisms to better understand gene function, or create commercially important cell lines and organisms.

Targeted Gene Correction

Efficient correction of deleterious changes in genes would provide researchers with the ability to fully understand the impact of the mutation on the organism, improve cell lines that are valuable research tools or have commercial importance, and of course correct genetic defects that can cause human disease. ZFN-mediated gene correction provides this capability across a wide range of organisms and cell types.

Since X-linked severe combined immunodeficiency disease (SCID) has been the target of gene therapy efforts, it was used as a model system to demonstrate the utility of ZFN-mediated gene correction.⁸ SCID is caused by mutations in the IL2R gene. Using ZFNs, targeted alterations were achieved in this gene in more than 18% of the recipient cells – without selection. Remarkably, about 7% of the cells acquired the genetic modification on both X chromosomes.

Successful genetic manipulation of an organism requires that progenitor and stem cells be modified efficiently ex vivo by gene transfer, with subsequent culturing and re-infusion into the organism to establish a stable and heritable change. This is of course a requirement for human gene therapy, and again SCID has been used as a model system to demonstrate the effectiveness of ZFN-mediated correction. Using a gene delivery approach based on integrase-defective lentiviral vectors, ZFNs have been used to generate permanent and heritable modifications of the IL2R gene in hematopoietic progenitor cells as well as human embryonic stem cell lines at high frequencies, allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.⁹ Up to 50% targeted gene addition was achieved in a panel of recipient human cell lines, and up to 5% of human embryonic stem cells. ZFN-mediated gene correction can clearly provide highly efficient gene correction that can be used in a wide variety of organisms.

Sangamo BioSciences: Pioneering Zinc Finger Research

Sangamo BioSciences has been a major leader in the development of zinc finger protein technologies as a means of genespecific editing. Currently, Sangamo is focused on using these technologies to develop gene regulation and correction therapeutics. They are currently conducting phase 1 and 2 trials for application of the up-regulation of endogenous VEGF-A in the treatment of diabetic neuropathy. Sangamo is also focusing on the development of therapeutics for cardiovascular and peripheral artery disease, cancer, neuropathic pain, HIV/AIDS, congestive heart failure and monogenic diseases.

Sangamo has led not only in the development of the design of zinc finger nucleases specific for essentially any known DNA sequence, but also in the development of applications of ZFNs for gene knockout, correction and gene integration in a wide variety of species, as well as gene editing in human stem cells.^4,5,7-9

Making ZFN Technology Accessible

In 2007, we formed an exclusive partnership with Sangamo to make ZFN technology easily available to research scientists around the world through its CompoZr™ brand line of products and services.

The CompoZr Custom ZFN offering, launched in Fall 2008, supplies partners with ZFNs that have been validated to target and edit customer-defined genes of interest. Deliverables of the CompoZr Custom ZFN service include ready-to-transfect amounts of ZFNs, as well as controls and reagents for screening for mutational events.

Future CompoZr products based on ZFN technology, available in 2009, will include ZFNs targeting popular genes, gene families, and pathways. These ‘off-the-shelf’ reagents will complement the CompoZr Custom ZFN offering and allow customers to easily edit genes in cell lines of interest.

To learn more about the CompoZr line of ZFN-based reagents, visit compozrzfn.com.

Conclusion

The ability to routinely edit genes in cell lines, animals and plants has long been a goal sought after by researchers seeking an in-depth understanding of complex genetic functions and applying that knowledge to develop new pharmaceuticals, improve agricultural productivity, and even cure human disease through gene therapy. Zinc finger nuclease technology promises to realize this goal by providing the tools to precisely and efficiently induce genetic changes in systems ranging from human stem cell lines to fruit flies, plants and a host of other organisms. Sangamo BioSciences has led the way in developing this revolutionary technology by designing ZFNs that are exquisitely specific and efficient, and demonstrating their utility for generating targeted gene knockouts, gene integration and even gene correction in relevant biological systems. Our ability to deliver high quality research reagents and services to researchers all over the world through the CompoZr brand of products, combined with Sangamo’s substantial expertise in the field of ZFNs, provide the life science research community with access to this unparalleled technology for genomic modification.