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Piggybac Transposase Tools for Genome Engineering.Pdf piggyBac transposase tools for genome engineering PNAS PLUS Xianghong Lia,b, Erin R. Burnightc, Ashley L. Cooneyd, Nirav Malanie, Troy Bradye, Jeffry D. Sanderf,g, Janice Staberd, Sarah J. Wheelanh, J. Keith Joungf,g, Paul B. McCray, Jr.c,d, Frederic D. Bushmane, Patrick L. Sinnd,1, and Nancy L. Craiga,b,1 aHoward Hughes Medical Institute and bDepartment of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; cGenetics Program, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242; dDepartment of Pediatrics, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242; eDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; fMolecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02129; gDepartment of Pathology, Harvard Medical School, Boston, MA 02115; and hDivision of Biostatistics and Bioinformatics, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Edited by Richard A. Young, Massachusetts Institute of Technology, Cambridge, MA, and approved May 1, 2013 (received for review December 30, 2012) The transposon piggyBac is being used increasingly for genetic stud- site-directed mutagenesis of the PB catalytic domain and isolated − ies. Here, we describe modified versions of piggyBac transposase an excision competent/integration defective (Exc+Int ) PB. We − that have potentially wide-ranging applications, such as reversible also find that introduction of the Exc+Int mutations into a hy- − transgenesis and modified targeting of insertions. piggyBac is dis- peractive version of PB (5, 8, 15) yields an Exc+Int transposase tinguished by its ability to excise precisely, restoring the donor site whose excision frequency is five- to six-fold higher than that of to its pretransposon state. This characteristic makes piggyBac useful wild-type PB. We speculate that the changed amino acids in the − for reversible transgenesis, a potentially valuable feature when gen- Exc+Int mutant are likely positions of interaction between the erating induced pluripotent stem cells without permanent altera- transposase and the target DNA. tions to genomic sequence. To avoid further genome modification Modification of nonviral vector systems to alter native in- following piggyBac excision by reintegration, we generated an ex- tegration patterns in a predictable manner would provide molec- + − cision competent/integration defective (Exc Int ) transposase. Our ular biological tools with relevance to diverse applications, including findings also suggest the position of a target DNA–transposase in- therapeutics for genetic disease and ex vivo stem cell manipulation. teraction. Another goal of genome engineering is to develop piggyBac inserts at target sites with the sequence TTAA but displays reagents that can guide transgenes to preferred genomic regions. little selectivity for particular regions of the genome other than Others have shown that piggyBac transposase can be active when + − a modest preference for regions of DNase I sensitivity (16, 17). A fused to a heterologous DNA-binding domain. An Exc Int trans- useful modificationofPBwouldbetobeabletoguideintegrations posase, the intrinsic targeting of which is defective, might also be to safe harbor sites. Others have shown that PB is distinguished by a useful intermediate in generating a transposase whose integra- its ability to remain active when fused to a DNA binding domain fi tion activity could be rescued and redirected by fusion to a site-speci c and that such fusions can bias insertion toward cognate sites (10, 16, DNA-binding domain. We show that fusion to two designed zinc fi fi − 18). Notably, Wilson and colleagues (19) fused a zinc nger protein nger proteins rescued the Int phenotype. Successful guided (ZFP) targeted to the upstream promoter region of the cell-cycle transgene integration into genomic DNA would have broad appli- − checkpoint kinase 2 protein-coding gene CHEK2 to the native cations to gene therapy and molecular genetics. Thus, an Exc+Int insect-derived piggyBac (iPB) transposase. The ZFP–iPB fusions transposase is a potentially useful reagent for genome engineering exhibited comparable transposition activity to unmodified trans- and provides insight into the mechanism of transposase–target posase and bound the target site in their study. However, no DNA interaction. fi GULOP | ROSA26 | protein-DNA interaction | Signi cance induced pluripotent stem cell production GENETICS DNA transposons that translocate by excision from a donor site NA “cut-and-paste” transposable elements are important tools and insertion into a target site are often used for genome Dfor genome engineering, such as insertional mutagenesis and engineering by insertional mutagenesis and transgenesis. The transgenesis. Research with the DNA transposon Sleeping Beauty, piggyBac element is especially useful because it can excise a “resurrected” transposon, has pioneered the use of DNA trans- precisely from an insertion site, restoring the site to its pre- transposon state. Precise excision is particularly useful when posons in mammalian cells (1, 2). piggyBac is also a DNA transposon transient transgenesis is needed, for example, in the transient and a promising alternative to Sleeping Beauty. piggyBac, originally introduction of transcription factors for induced pluripotent stem isolated from the cabbage looper moth Trichoplusia ni genome (3), cell production. We have used mutagenesis to generate an Exci- has a large cargo size (4), is highly active in many cell types, and + − – sion Integration transposase that allows piggyBac excision mediateslong-termexpressioninmammaliancellsinvivo(510). without potentially harmful reintegration. These mutations likely piggyBac is also distinguished by its ability to excise precisely (11), lie in a target DNA-binding domain. thus restoring the donor site to its pretransposon insertion sequence. Because it can excise precisely, piggyBac is especially useful if Author contributions: X.L., E.R.B., P.B.M., P.L.S., and N.L.C. designed research; X.L., E.R.B., a transgene is only transiently required. Transient integration and and A.L.C. performed research; J.D.S. contributed new reagents/analytic tools; X.L., N.M., expression of transcription factors are important approaches to T.B., J.S., S.J.W., J.K.J., P.B.M., F.D.B., P.L.S., and N.L.C. analyzed data; and X.L., F.D.B., generate transgene-free induced pluripotent stem cells (iPSCs) P.L.S., and N.L.C. wrote the paper. fl fi (12, 13) as well as directed differentiation of specific cell types for Con ict of interest statement: J.K.J. has a nancial interest in Transposagen Biopharma- ceuticals. J.K.J.’s interests were reviewed and are managed by Massachusetts General both research and clinical use. Removal of the transgenes is key for Hospital and Partners HealthCare in accordance with their conflict of interest policies. potential therapeutic applications of iPSCs. piggyBac has been This article is a PNAS Direct Submission. used as a vector for reversible integration; however, reintegration Freely available online through the PNAS open access option. of the transposon catalyzed by piggyBac (PB) transposase occurs in 1 To whom correspondence may be addressed. E-mail: [email protected] or patrick-sinn@ 40–50% of cells (14). To generate iPSCs without any genetic uiowa.edu. change, a PB mutant, which can promote only excision (Exc+)and − This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. not integration (Int ), would be a useful tool. Here we performed 1073/pnas.1305987110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1305987110 PNAS | Published online May 30, 2013 | E2279–E2287 integration events were recovered at the native site in HEK293 (Int+) (Fig. 2C). The single mutants R372A or K375A are also cells, presumably due to the paucity of TTAA sites within the Int+ (Fig. S2). In contrast, transposase mutants R245A, R275A/ target region. Indeed, this hypothesis is supported with subsequent R277A, S351E, R372A/K375A, and R388A had low integration experiments demonstrating a significant increase in integration activity similar to the no transposase (−Tps) control (Fig. 2C), − recovery near a CHEK2 target site engineered with 12 TTAA indicating that they are integration-defective (Int ). Thus, these − sites. Stringent target-site selection in the genome has not been five mutants are categorized as candidate Exc+Int mutants. demonstrated with any transposon system, including piggyBac or Sleeping Beauty (20). In Vitro Analysis of the Exc+Int− Candidates. To further analyze the − In this study we analyzed the effects of targeting by the PB Exc+Int candidates, we assayed their transposition activities − Exc+Int PB upon fusion to several ZFPs designed to recognize in a cell-free assay using transposase purified from Escherichia putative safe harbor regions. Importantly, we observed that coli (22). piggyBac transposition, starting with excision, initiates N-terminal fusion of heterologous ZFPs rescued the integration with a nick at the 3′ end of the transposon, followed by the attack defect. No significant targeting to the ZFP-binding sites, how- of the newly exposed end on the complementary strand, four + − ever, was observed. We conclude that an Exc Int piggyBac nucleotides within the donor DNA that releases the flanking transposase provides a tool for
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