Article

Site-directed transposon integration in human cells

Division of General Pediatrics, Stanford University, Palo Alto, California, United States
Nucleic Acids Research (Impact Factor: 9.11). 02/2007; 35(7):e50. DOI: 10.1093/nar/gkm089
Source: PubMed

ABSTRACT

The Sleeping Beauty (SB) transposon is a promising gene transfer vector that integrates nonspecifically into host cell genomes. Herein, we attempt
to direct transposon integration into predetermined DNA sites by coupling a site-specific DNA-binding domain (DBD) to the
SB transposase. We engineered fusion proteins comprised of a hyperactive SB transposase (HSB5) joined via a variable-length linker to either end of the polydactyl zinc-finger protein E2C, which binds
a unique sequence on human chromosome 17. Although DBD linkage to the C-terminus of SB abolished activity in a human cell transposition assay, the N-terminal addition of the E2C or Gal4 DBD did not. Molecular
analyses indicated that these DBD-SB fusion proteins retained DNA-binding specificity for their respective substrate molecules
and were capable of mediating bona fide transposition reactions. We also characterized transposon integrations in the presence of the E2C-SB fusion protein to determine
its potential to target predefined DNA sites. Our results indicate that fusion protein-mediated tethering can effectively
redirect transposon insertion site selection in human cells, but suggest that stable docking of integration complexes may
also partially interfere with the cut-and-paste mechanism. These findings illustrate the feasibility of directed transposon
integration and highlight potential means for future development.

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    • "Additionally, transposases catalyze the rearrangement of endogenous elements and have been used for artificial genome manipulation (Ivics et al. 2009). Targeting of transposase activity with ZF, TALE, or CRISPR/Cas scaffolds is also an active area of research (Yant et al. 2007; Voigt et al. 2012; Galvan et al. 2014). Importantly , targeted recombination or transposition may reduce cellular toxicity relative to the introduction of DSBs and their subsequent resolution through NHEJ or HDR. "
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    ABSTRACT: Advances in genome engineering technologies have made the precise control over genome sequence and regulation possible across a variety of disciplines. These tools can expand our understanding of fundamental biological processes and create new opportunities for therapeutic designs. The rapid evolution of these methods has also catalyzed a new era of genomics that includes multiple approaches to functionally characterize and manipulate the regulation of genomic information. Here, we review the recent advances of the most widely adopted genome engineering platforms and their application to functional genomics. This includes engineered zinc finger proteins, TALEs/TALENs, and the CRISPR/Cas9 system as nucleases for genome editing, transcription factors for epigenome editing, and other emerging applications. We also present current and potential future applications of these tools, as well as their current limitations and areas for future advances.
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    • "In the presence of transposase supplied in trans, any gene of interest flanked by inverted repeats (IRs) represents a substrate for transposition resulting in somatic integration into a TA-dinucleotide [8,10]. Very recently hyperactive SB transposase versions HSB5 [11] and SB100X [12] were generated by mutagenesis screens which resulted in 10- and 100-fold increased integration efficiencies, respectively. Previous data suggest that the target sites of integration after SB mediated recombination show a close to random genomic distribution profile. "
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    ABSTRACT: Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells.
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    • "Targetable transposition, using chimeric proteins consisting of a DBD fused to a transposase, can be used to preferentially insert transgenes near a specific sequence. A variety of DBDs have been used to bias transposon integration on recipient plasmids in various cell types (28,42–49). Recently, endogenous transpositional targeting has been achieved (28,42). "
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    ABSTRACT: Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.
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