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July 01, 2010 (Vol. 30, No. 13)

Zinc Finger Nucleases for Genome Editing

ZFN Technology Is Being Used to Facilitate the Genetic Engineering of Mammalian Cells

  • Click Image To Enlarge +
    Figure 1. A zinc finger nuclease (ZFN) is composed of two functional domains: (A) an engineered zinc finger protein that binds the target DNA and (B) the type II restriction endonuclease FokI.

    For decades, mammalian genomes have remained resistant to targeted modification and researchers have generally lacked robust tools for efficient and routine mutagenesis. This is not surprising as mammalian genomes are littered with large amounts of repetitive elements, some of which constitute ~21% of the entire genome.

    If the human genome were more permissive to homologous recombination, the stability of the genome would be largely compromised. Thus, in light of this and other protective mechanisms mammalian cells have established for genome stability, the genetic engineering of mammalian cells has remained difficult.

    In cell types where homologous recombination is naturally suppressed, methods have been developed to induce hyper-recombinant phenotypes. For example, phage proteins have been used to globally increase the rate of homologous recombination in Escherichia coli and Mycobacterium tuberculosis.

    This technological advancement facilitated the rapid generation of a complete, genome-wide knockout library of E. coli strains, each lacking a specific gene. However, application of this or similar technologies is impractical in mammalian cells since a global increase in recombination would destabilize the genome through random recombination of repetitive elements. Indeed, such genomic instability is known to be associated with many human diseases.

    The recent development of engineered zinc finger nucleases (ZFNs) provides researchers with a technological solution to mammalian genome editing. ZFNs can be designed to generate site-specific double-strand breaks (DSBs), which are repaired by either nonhomologous end joining (NHEJ), leading to gene knockout  or homologous recombination (HR), achieving precise gene replacement, insertion, or deletion (Figure 1).

    In this way, gene editing becomes possible in a localized area without compromising the stability of the entire genome. Consequently, in the area surrounding a ZFN-mediated DSB, the rate of DNA repair can be elevated by several orders of magnitude, often increasing mutagenic frequencies from 10-6–10-5 up to 10-2 without global changes in genomic stability.

    Such a large increase in efficiency has enabled the direct screening of clonal cells for mutations in convenient 96-well formats, without relying on antibiotic selection markers. The freedom from using antibiotic selection enables a new level of “surgical” genome modification such as the mutation of codons and the modification of single nucleotide polymorphisms (SNPs).

  • Making Delicate Point Mutations

    In the decades since the development of recombinant DNA technology, the exploration of gene function has largely relied on creating the desired mutations to protein coding sequences as cDNA harbored on exogenous plasmids. The modified genes are then shuttled into human cells and expressed from exogenous promoters.

    Endogenous gene expression, however, occurs in a much more complex environment, reliant on exon-intron splicing and many other gene regulation factors mediated by interactions of 5´-UTR and 3´-UTR regions with control elements such as transcription factors and microRNAs. Additionally, the sheer size of many genes (20–100 kb and higher) makes it cumbersome to isolate and express them on exogenous plasmids with their exon, intron, and UTR structures intact.

    The creation of targeted point mutations at specific positions on the mammalian chromosome has proven to be a difficult task, even when homologous recombination rates are elevated in specific cell types. ZFNs enable delicate, specific mutagenesis of single base sites in the native context of the gene, lending more biological relevance to the resulting data generated by targeted mutagenesis.

    The ability to make such mutations enables a more rigorous investigation of SNPs, in which single base pair differences are associated with disease states. ZFN-mediated single base pair mutations have been accomplished at several different genomic loci using ZFNs in conjunction with a donor DNA containing the desired mutation.

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