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Genome-Wide Target Profiling of Piggybac and Tol2 in HEK

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Genome-Wide Target Profiling of Piggybac and Tol2 in HEK Meir et al. BMC Biotechnology 2011, 11:28 http://www.biomedcentral.com/1472-6750/11/28 RESEARCHARTICLE Open Access Genome-wide target profiling of piggyBac and Tol2 in HEK 293: pros and cons for gene discovery and gene therapy Yaa-Jyuhn J Meir1*, Matthew T Weirauch2, Herng-Shing Yang3, Pei-Cheng Chung4,RobertKYu5 and Sareina C-Y Wu4* Abstract Background: DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side- by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery. Results: We have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3’-and 67 bp 5’-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2. Conclusions: The non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting. Background transgenesis and insertional mutagenesis [2-7]. Such DNA transposons are natural genetic elements residing tools, however, have not been available for genome in the genome as repetitive sequences. A simple trans- manipulations in vertebrates or mammals until the reac- poson is organized by terminal repeat domains (TRDs) tivation of a Tc1/mariner-like element, Sleeping Beauty, embracing a gene encoding a catalytic protein, transpo- from fossils in the salmonid fish genome [8]. Since its sase, required for its relocation in the genome through a awakening, Sleeping Beauty has been used as a tool for “cut-and-paste” mechanism. Since the first discovery of versatile genetic applications ranging from transgenesis DNA transposons in Maize by Barbara#McClintock in to functional genomics and gene therapy in vertebrates 1950 [1], transposons have been used extensively as including fish, frogs, mice, rats and humans [9]. Subse- genetic tools in invertebrates and in plants for quently, naturally existing transposons, such as Tol2 and piggyBac, have also been shown to effectively transpose in vertebrates. * Correspondence: [email protected]; [email protected] The Medaka fish (Orizyas latipes) Tol2,belongingto 1Department of Biomedical Sciences, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan the hAT family of transposons, is the first known natu- 4Molecular Medicine Research Center, Chang Gung University, 259 Wen-Hwa rally occurring active DNA transposon discovered in 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan vertebrate genomes [10]. Tol2 is a standard tool for Full list of author information is available at the end of the article © 2011 Meir et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Meir et al. BMC Biotechnology 2011, 11:28 Page 2 of 19 http://www.biomedcentral.com/1472-6750/11/28 manipulating zebrafish genomes and has been demon- specific targeting of therapeutic genes due to a robust strated to transpose effectively in frog, chicken, mouse enzymatic activity of the piggyBac transposase and flex- and human cells as well [11]. Recent studies found that ibility the transposase displays towards molecular engi- Tol2 is an effective tool both for transgenesis via pro- neering. Finally, results of our in-depth analyses of nuclear microinjection and germline insertional muta- piggyBac target sequences highlight the need to first genesis in mice [12]. Cabbage looper moth (Trichoplusia scrutinize the piggyBac favored target sites for the thera- ni) piggyBac is the founder of the piggyBac superfamily peutic cell type of interest before designing a custo- and is widely used for mutagenesis and transgenesis in mized DNA binding protein for fusing with the insects [13]. Recently, piggyBac was shown to be highly piggyBac transposase to achieve site-specific therapeutic active in mouse and human cells and has emerged as a gene targeting. promising vector system for chromosomal integration, including insertional mutagenesis in mice and nuclear Results reprogramming of mouse fibroblasts to induced-pluripo- Transposition activity of piggyBac and Tol2 in mammalian tent stem cells [14-19]. cells To date, most gene therapy trials have utilized viral With the ultimate goal of identifying and targeting safe vectors for permanent gene transfer due to their high sites in the genome at which to insert corrective genes, transduction rate and their ability to integrate therapeu- we previously explored three active mammalian transpo- tic genes into host genomes for stable expression. How- sases, piggyBac, Tol2 and SB11 (a hyperactive Sleeping ever, serious problems associated with most viral Beauty) for their sensitivity to molecular modification vectors, such as limited cargo capacity, host immune [15]. After fusing the GAL4 DNA binding domain to response, and oncogenic insertions (as evidenced by the the N-terminus of the three transposases, we only retrovirus-based gene therapy) highlight an urgent need detected a slight change in the activity of the piggyBac for developing effective non-viral therapeutic gene deliv- transposase, whereas the same modification nearly abol- ery systems [20,21]. Recently, Sleeping Beauty, Tol2, and ished the activity of Tol2 and SB11 [15]. A recent piggyBac transposon-based vector systems have been genetic screen has yielded a novel hyperactive Sleeping explored for their potential use in gene therapy with Beauty transposase (designated as SB100X) that was proven successes [22-25]. However, for therapeutic pur- shown to be more active than piggyBac under restrictive poses, a large cargo capacity is often required. The conditions that support their peak activity [28]. How- transposition efficiency of Sleeping Beauty is reduced in ever, in this study we chose to focus on piggyBac and a size-dependent manner with 50% reduction in its Tol2 but not Sleeping Beauty for the following reasons: activity when the size of the transposon reaches 6 kb (1) all of the reported attempts to modify the SB11 [26]. Tol2 and piggyBac, however, are able to integrate transposase either N- or C-terminally result in a com- up to 10 and 9.1 kb of foreign DNA into the host gen- plete elimination or a significant reduction in transpo- ome, respectively, without a significant reduction in sase activity; (2) Sleeping Beauty is more susceptible to their transposition activity [14,22]. Additionally, by a over expression inhibition than piggyBac and Tol2;(3) direct comparison, we have observed that Tol2 and pig- the cargo capacity of Sleeping Beauty is limited; and (4) gyBac are highly active in all mammalian cell types unlike Tol2 and piggyBac that are active in all mamma- tested, unlike SB11 (a hyperactive Sleeping Beauty), lian cell types tested, Sleeping Beauty display cell-type which exhibits a moderate and tissue-dependent activity dependent activity [15,27,34]. [15]. Because of their high cargo capacity and high We have demonstrated that piggyBac and Tol2 display transposition activity in a broad range of vertebrate cell high transposition activity in several cell lines [15]. We types, piggyBac and Tol2 are two promising tools for now wish to explore the possibility of further enhancing basic genetic studies and preclinical experimentation. their activity by trimming non-essential sequences from Our goal here was to evaluate the pros and cons of pig- both transposons. Using a PCR-based strategy we gener- gyBac and Tol2 for the use in gene therapy and gene ated pPB-cassette3short with the shortest TRDs discovery by performing a side-by-side comparison of reported replacing the long ones of the pXLBacII-cas- both transposon systems. In this study, we reported for sette (Figure 1A) [29,30]. Similarly, based on the pre- the first time the identification of the shortest effective vious report, a new Tol2 donor, pTol2mini-cassette, piggyBac TRDs as well as several piggyBac and Tol2 hot- with minimal terminal repeats [31] replacing the long spots. We also observed that piggyBac and Tol2 display ones of Tol2ends-cassette was also constructed (Figure non-overlapping targeting preferences, which makes 1A). The new helper plasmids of piggyBac (pPRIG-piggy- them complementary
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