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Zinc-Finger Nucleases: Expanding the Gene Editor's Tool Kit

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Zinc finger nucleases (ZFNs) have become useful reagents when it comes to manipulating the genomes in plants and animals. As a result, they are becoming increasingly widespread throughout academic campuses and industrial applications worldwide.

 Zinc finger nucleases (ZFNs) have become useful reagents when it comes to manipulating the genomes in plants and animals. As a result, they are becoming increasingly widespread throughout academic campuses and industrial applications worldwide. ZFNs are useful in their ability to recognize and bind to a 3-to 4-bp DNA sequence, but are unique in their ability to bind an extended nucleotide sequence typically between 9 bp to 18 bp in length (or 3x bigger) on the complimentary strand of DNA [1]. For this reason, ZFNs function similarly to restriction digest enzymes, but are classified separetly as fusion proteins.

 Originally adapted from bacterial transcription factors containing zinc-finger binding motifs, ZFNs have been modified to attach to the endonuclease domain of the bacterial restriction enzyme, FokI. Moreover, ZFNs are designed as a pair in order to recognize both site-specific sequences which flank the site of interest, one on the 5-to-3 forward strand and the other on the 3-to-5 reverse strand. Once the pair of FokI domains bind to both sides of the specified site, the ZFN dimerizes and cleaves the DNA at the location, generating a double-strand break (DSB) with 5' overhangs [2]. zinc.finger_nucleases  

Image courtesy of: Nature Reviews. ®2010.

Engineering Delicate Point Mutations

Despite the many advantages of using ZFNs in genome editing, there are several potential disadvantages. There is not yet a reliable method to engineer new ZFNs, since it requires a bit of sophistication to link together multiple zinc-finger domains capable of binding to an extended stretch of nucleotides with high affinity [3]. Another potential disadvantage is that selecting a target site is limited since the ZFN apparatus can only be used to target binding sites every 200 bps in DNA sequences. (Note: some commercial sources of ZFNs offer higher design densities, with the ability to target binding sites every 50 bps in DNA sequences.) While this may be a nonissue if a clinical investigator seeks to knock out a gene, since a translational frameshift introduced anywhere in the early coding sequence of the gene can produce the desired result, but could present a challenge if a particular site is required (e.g. to knock in a specific mutation) [4]. So, to circumvent some of these challenges, researchers are developing an academic consortium, a kind of open-source database chalk full of zinc finger components and protocols to perform screens necessary to identify ZFNs that bind with high affinity to a desired sequence [5,6]. Since the introduction of this open-source database, alternative platforms to engineer optimized ZFNs have emerged, each with varying degrees of speed, flexibility in site selection, and success rates [7]. Nonetheless, it could still take weeks to months for specialists to obtain optimized ZFNs. Finally, about the use of proteins designed to introduce DSBs into the genome of an organism raises significant concerns that they will do so not only at the desired site but also at off-target sites, as well. One study in which ZFNs were used for genome editing in human pluripotent stem cells, the investigators identified a total of ten possible off-target genomic sites based on high-sequence similarity to the on-target site and found a single off-target mutation out of the 184 clones assessed [8]. Two other subsequent studies seeking to identify potential off-target sites for several ZFN pairs revealed off-target events at numerous motifs in a cultured human tumor cell lines [9,10]. Thus, investigators should be cognizant of this possibility that ZFNs designed for a particular purpose may incur undesired off-target events at a low rate. One strategy that has emerged to reduce off-target events is to use a pair of ZFNs that have distinct FokI domains that are obligate heterodimers [11,12,13]. This prevents a single ZFN from binding to two adjacent off-target sites and generating a DSB; rather, the only way an off-target event could occur is if both ZFNs in a pair bind adjacently and thus allow the FokI dimer to form. Another strategy that has been demonstrated to reduce off-target events is the introduction of purified ZFN proteins into cells [14]. In spite of that, zinc fingers have proven to be pretty revolutionary.  Most famously, they have played a role in one of the most awesome biological advances in our lifetimes to date: stem cell gene therapy for HIV. Also the ZFN plasmid-based approach for gene therapy holds tremendous potential to circumvent all the problems associated with the viral delivery of therapeutic genes [15]. So there is still a bit of trial and error and you can't always target the exact sequence you want to and you might need to settle for another site a little ways away from your ideal site.  

Regulatory Framework for GE Materials

All genetically engineered (GE) materials, including GE foods and pharmaceuticals, are regulated by a matrix of federal law and the Coordinated Framework for Regulation of Biotechnology, established in 1986, by the Office of Science and Technology Policy (OSTP). This established a means for coordination among federal agencies to regulate GE plants, organisms, and products made using GE plants, organisms, or materials. We have to start regulating by the properties of the crop, not the techniques by which it was modified, which is what we're doing now, says Nina Fedoroff, former science and technology advisor to the US Secretary of State. Ironically, the next generation of gene-editing technologies may sidestep the regulatory process entirely. The USDA regulates transgenic crops through the Plant Protection Act, which gives it the power to rule on genetic parts that come specifically from plant pests. Zinc-finger nucleases (ZFNs), for example, don't originate from pests, and, therefore, appear to fall outside the regulatory framework. In 2009, Vipula Shukla at Dow AgroSciences used ZFNs to produce herbicide resistance in corn without adding any foreign genes [16]. Presumably, these seeds will be treated like any conventional breed. Because the changes you introduce by those techniques are exactly like those you can make by classical mutagenesis, it shouldn't be subject to this horrendous regulation, Fedoroff says. Similar constructs called transcription activator like effector nucleases (TALENs) could make gene editing even easier, they originate from the plant pest Xanthomonas, and might be captured under the current framework. The USDAs Animal and Plant Health Inspection Service (APHIS) has yet to decide on its role in the process. APHIS is currently considering the regulatory status of zinc-finger nucleases and transcription activator like effector nucleases, says spokesman Richard Bell. The decision has the potential to change the entire industry.   JOURNAL REFERENCES [1,2,4] Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. "Genome editing with engineered zinc finger nucleases." Nature Review Genetics. 2010; 11(9):636-646. [3] Ramirez CL, et al. "Unexpected failure rates for modular assembly of engineered zinc fingers." Nature Methods. 2008; 5(5):374-375. [5] Maeder ML, et al. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell. 2008; 31(2):294-301. [6] Maeder ML, Thibodeau-Beganny S, Sander JD, Voytas DF, Joung JK. "Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays." Nature Protocols. 2009; 4(10):1471-1501. [7]  Gupta A, Christensen RG, Rayla AL, Lakshmanan A, Stormo GD, Wolfe SA. An optimized two-finger archive for ZFN-mediated gene targeting. Nat Methods. 2012; 9(6):588-590. [8]  Hockemeyer D, et al. "Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases." Nature Biotechnology. 2009; 27(9):851-857. [9] Gabriel R, et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol. 2011; 29(9):816-823. [10] Pattanayak V, Ramirez CL, Joung JK, Liu DR. "Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection." Nature Methods. 2011; 8(9):765-770. [11] Doyon Y, et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods. 2011; 8(1):74-79. [12] Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T. "Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases." Nature Biotechnology. 2007; 25(7):786-793. [13] Miller JC, et al. "An improved zinc-finger nuclease architecture for highly specific genome editing." Nature Biotechnology. 2007; 25(7):778-785. [14] Gaj T, Guo J, Kato Y, Sirk SJ, Barbas CF. Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods. 2012; 9(8):805-807. [15] Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK. "FLASH assembly of TALENs for high-throughput genome editing." Nature Biotechnology. 2012; 30(5):460-465. [16] Gruskin D. "Agbiotech 2.0." Nature Biotechnology. 2012; 30:211-214.