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Mar 1, 2014 (Vol. 34, No. 5)

Comparing Genome Editing Technologies

  • Click Image To Enlarge +
    Genome editing technologies also have applications in the regulation of gene expression and the manipulation of the epigenome. [Laguna Design/Science Source]

    The Genomic Revolution has promised to advance medicine and biotechnology by providing scientists with enormous amounts of data that can be converted into useful information.

    Over 10 years ago, the Human Genome Project produced the first draft of the more than 3 billion base pairs of DNA that make up the genetic code in each of our cells.

    More recent efforts like the 1000 Genomes and HapMap Projects have since focused on identifying the differences within these billions of base pairs of DNA between individuals, while genome-wide association studies have pinpointed specific sequences that determine health and disease. The ENCODE Project and other studies have annotated chromatin states, regulatory elements, transcription factor binding sites, and other epigenetic states throughout the genome.

    Dozens of other species have since undergone similar analyses, with the number of sequenced genomes continuously growing. Collectively, these efforts have generated an incredibly rich source of data that promises to aid our understanding of the function and evolution of any genome. However, until recently, scientists have been lacking the tools necessary to interrogate the structure and function of these elements.

    While conventional genetic engineering methods could be used to add extra genes to cells, they cannot be easily used to modify the sequences or control the expression of genes that already exist within these genomes. These types of tools are necessary to determine not only the function of genes, but also the role of genetic variants and regulatory elements. They can also be used to overcome longstanding challenges in the field of gene therapy. Without these technologies, it has been difficult—and in some cases impossible—for scientists to capitalize on the Genomic Revolution.

    Genome editing technologies also have applications in the regulation of gene expression and the manipulation 
    of the epigenome.  
    Laguna Design/Science Source
  • Genome Editing

    Click Image To Enlarge +
    Figure 1. The structure and mode of DNA recognition of zinc finger nucleases, TALE nucleases, and CRISPR/Cas9 [Duke University]

    A potential route for introducing precise changes into the genome was suggested by the discovery of homologous recombination and its application in creating transgenic and knockout mice, for which the Nobel Prize in Physiology or Medicine was awarded in 2007. This method, however, was only efficient in mouse embryonic stem cells and not immediately applicable to cells from other species or even other mouse cell types.

    But in 1994 a breakthrough study showed that creating a double-strand break at a specific site in the genome could stimulate the cellular DNA repair pathway and increase the frequency of homologous recombination at the break point by many orders of magnitude. Critically, this study also suggested a direct approach for editing virtually any gene sequence in many different cell types and species using homologous recombination—but only if DNA breaks could be targeted to specific sequences.

    The re-engineering of molecular machinery to recognize new sequences in complex genomes is a daunting task. However, in 1991, the crystal structure of Zif268, a naturally occurring zinc finger protein, provided insight into how Nature solved this problem. This structure led to the discovery that zinc finger proteins, among the most common class of DNA-binding proteins across all domains of life, recognize DNA using independent and modular domains that make specific contacts with three base pairs of DNA (Figure 1). This work suggested that these domains could be redesigned to recognize new base pair combinations and linked together to form new proteins.

    Subsequent research by several laboratories led to the development of technologies for making custom synthetic zinc finger proteins that can be targeted to a broad range of sites in almost any genome. This constituted the first technology for targeting and regulating specific endogenous genes.

    In the late-1990s, the catalytic domain of the FokI endonuclease, which nonspecifically cleaves DNA, was fused to custom zinc finger proteins to generate the first zinc finger nuclease (ZFN). Because the DNA-binding specificity of zinc finger proteins could be reprogrammed, new ZFNs could be rapidly formed and used to introduce targeted double-strand breaks to almost any gene in the genome.

    Critically, because FokI acts as a dimer, it’s necessary to engineer two ZFNs that target opposite strands of DNA in a head-to-head configuration (Figure 1). Thus, when two FokI catalytic domains assemble together at the targeted DNA site, a double-strand break is created and genome editing is initiated.

  • New and Improved Tools

    Despite the powerful potential of ZFN-mediated genome editing and the many notable achievements made using this technology, the scientific community was slow to broadly adopt it. This was largely due to the technical expertise necessary to engineer new zinc finger proteins, as well as the need to screen many ZFNs in order to uncover enzymes with high activity and low toxicity. Arguably the lack of cheap and widespread DNA synthesis and public plasmid repositories in the early 2000s also slowed the development of this technology.

    But in 2009, the DNA recognition code of another modular DNA-binding protein–transcription activator-like effectors (TALEs)–was reported. TALEs are proteins produced by plant pathogenic bacteria and, in contrast to zinc fingers, recognize a single base pair of DNA using only a single protein module (Figure 1). Therefore, new TALE proteins capable of recognizing almost any DNA sequence could be assembled from only four pieces.

    Within two years of these first reports, several groups developed methods to quickly and economically assemble DNA sequences encoding new TALE proteins, as well as methods for fusing them to the FokI catalytic domain to create TALE nucleases, or TALENs. Most remarkably, almost every new report agreed that the majority of assembled TALENs were highly active at their intended target sites, in contrast to ZFNs, which often required screening or selection methods.

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