The <Emphasis Type="Italic">piggyBac</Emphasis> Transposon as a Tool in Genetic Engineering

Transposons are mobile genetic elements that are part of the genomic DNA of numerous organisms and belong to two classes. Unlike class I transposons, class II DNA transposons do not use the stage of RNA synthesis in their transition; they perform it by the cut-and-paste mechanism or with a replicative transposition. The integration of a DNA transposon in a new site results in the duplication of a target sequence on either side of a transposon, and its excision is, as a rule, associated with insertions and deletions. The piggyBac transposon isolated from the Trichoplusia ni moth differs from other mobile elements of its class. Due to its unique ability to leave no traces after excision from an insertion site and to perform successful transposition and transference of large DNA fragments, piggyBac is a convenient tool for the development of gene engineering approaches. The TTAA sequence serves as a target site for transposon integration: insertion in the AT-rich DNA regions is more frequent. The ability of piggyBac to be transferred to a new area independently of the cell apparatus and to restore a DNA site without error after excision lies in the mechanism of its transposition, which is discussed in detail in the present review. Along with other transposons and viruses, the piggyBac transposon is widely used in the transgenesis of various organisms; it also finds application in insertion mutagenesis and gene therapy.     

Cognate restriction of transposition by piggyBac-like proteins

Mobile genetic elements have been harnessed for gene transfer for a wide variety of applications including generation of stable cell lines, recombinant protein production, creation of transgenic animals, and engineering cell and gene therapy products. The piggyBac transposon family includes transposase or transposase-like proteins from a variety of species including insect, bat and human. Recently, human piggyBac transposable element derived 5 (PGBD5) protein was reported to be able to transpose piggyBac transposons in human cells raising possible safety concerns for piggyBac-mediated gene transfer applications. We evaluated three piggyBac-like proteins across species including piggyBac (insect), piggyBat (bat) and PGBD5 (human) for their ability to mobilize piggyBac transposons in human cells. We observed a lack of cross-species transposition activity. piggyBac and piggyBat activity was restricted to their cognate transposons. PGBD5 was unable to mobilize piggyBac transposons based on excision, colony count and plasmid rescue analysis, and it was unable to bind piggyBac terminal repeats. Within the piggyBac family, we observed a lack of cross-species activity and found that PGBD5 was unable to bind, excise or integrate piggyBac transposons in human cells. Transposition activity appears restricted within species within the piggyBac family of mobile genetic elements.     

The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ∼45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.     

Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System

Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors.     

A benchmark and an algorithm for detecting germline transposon insertions and measuring de novo transposon insertion frequencies

Transposons are genomic parasites, and their new insertions can cause instability and spur the evolution of their host genomes. Rapid accumulation of short-read whole-genome sequencing data provides a great opportunity for studying new transposon insertions and their impacts on the host genome. Although many algorithms are available for detecting transposon insertions, the task remains challenging and existing tools are not designed for identifying de novo insertions. Here, we present a new benchmark fly dataset based on PacBio long-read sequencing and a new method TEMP2 for detecting germline insertions and measuring de novo ‘singleton’ insertion frequencies in eukaryotic genomes. TEMP2 achieves high sensitivity and precision for detecting germline insertions when compared with existing tools using both simulated data in fly and experimental data in fly and human. Furthermore, TEMP2 can accurately assess the frequencies of de novo transposon insertions even with high levels of chimeric reads in simulated datasets; such chimeric reads often occur during the construction of short-read sequencing libraries. By applying TEMP2 to published data on hybrid dysgenic flies inflicted by de-repressed P-elements, we confirmed the continuous new insertions of P-elements in dysgenic offspring before they regain piRNAs for P-element repression. TEMP2 is freely available at Github: https://github.com/weng-lab/TEMP2.     

Precise marker excision system using an animal‐derived piggyBac transposon in plants

Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth‐derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase‐expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T₀ plants without concomitant re‐integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection.     

Interplasmid transposition demonstrates piggyBac mobility in vertebrate species

The piggyBac transposon is an extremely versatile helper-dependent vector for gene transfer and germ line transformation in a wide range of invertebrate species. Analyses of genome sequencing databases have identified piggyBac homologues among several sequenced animal genomes, including the human genome. In this report we demonstrate that this insect transposon is capable of transposition in primate cells and embryos of the zebrafish, Danio rerio. piggyBac mobility was demonstrated using an interplasmid transposition assay that has consistently predicted the germ line transformation capabilities of this mobile element in several other species. Both transfected COS-7 primate cells and injected zebrafish embryos supported the helper-dependent movement of tagged piggyBac element between plasmids in the characteristic cut-and-paste, TTAA target-site specific manner. These results validate piggyBac as a valuable tool for genetic analysis of vertebrates.     

A long terminal repeat retrotransposon of fission yeast has strong preferences for specific sites of insertion

The successful dispersal of transposons depends on the critical balance between the fitness of the host and the ability of the transposon to insert into the host genome. One method transposons may use to avoid the disruption of coding sequences is to target integration into safe havens. We explored the interaction between the long terminal repeat retrotransposon Tf1 and the genome of the yeast Schizosaccharomyces pombe. Using techniques that were specifically designed to detect integration of Tf1 throughout the genome and to avoid bias in this detection, we generated 51 insertion events. Although 60.2% of the genome of S. pombe is coding sequence, all but one of the insertions occurred in intergenic regions. We also found that Tf1 was significantly more likely to insert into intergenic regions that included polymerase II promoters than into regions between convergent gene pairs. Interestingly, 8 of the 51 insertion sites were isolated multiple times from genetically independent cultures. This result suggests that specific sites in intergenic regions are targeted by Tf1. Perhaps the most surprising observation was that per kilobase of nonrepetitive sequence, Tf1 was significantly more likely to insert into chromosome 3 than into one of the other two chromosomes. This preference was found not to be due to differences in the distribution or composition of intergenic sequences within the three chromosomes.     

In Vivo Himar1 Transposon Mutagenesis of Burkholderia pseudomallei

Burkholderia psedudomallei is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a Himar1-based random mutagenesis system for B. pseudomallei (HimarBP). The transposons contain a Flp recombinase-excisable, approved kanamycin resistance selection marker and an R6K origin of replication for transposon rescue. In vivo mutagenesis of virulent B. pseudomallei strain 1026b was highly efficient, with up to 44% of cells transformed with the delivery plasmid harboring chromosomal HimarBP insertions. Southern analyses revealed single insertions with no evidence of delivery plasmid maintenance. Sequence analysis of rescued HimarBP insertions revealed random insertions on both chromosomes within open reading frames and intergenic regions and that the orientation of insertions was largely unbiased. Auxotrophic mutants were obtained at a frequency of 0.72%, and nutritional supplementation experiments supported the functional assignment of genes within the respective biosynthetic pathways. HimarBP insertions were stable in the absence of selection and could be readily transferred between naturally transformable strains. Experiments with B. thailandensis suggest that the newly developed HimarBP transposons can also be used for random mutagenesis of other Burkholderia spp., especially the closely related species B. mallei. Our results demonstrate that comprehensive transposon libraries of B. pseudomallei can be generated, providing additional tools for the study of the biology, pathogenesis, and antibiotic resistance of this pathogen.     

Comparison of piggyBac transposition efficiency between linear and circular donor vectors in mammalian cells

The piggyBac transposon has recently attracted attention as a tool for transgene integration in mammalian cells. However, previous studies involving piggyBac investigated only transposition from circular DNA, although some linear DNA vectors are used to transfect mammalian cells. In this study, we compared the transposition efficiency of piggyBac between linear and circular DNA. Colony counting assay, luciferase assay, and plasmid rescue assay showed that piggyBac transposon can transpose from linear DNA, but its efficiency is lower than the transposition efficiency from circular DNA. These results suggest that circular DNA is more suitable as donor vectors of piggyBac than linear DNA.     

IPB7 transposase behavior in Drosophila melanogaster and Aedes aegypti

Transposons are used in insect science as genetic tools that enable the transformation of insects and the identification and isolation of genes though their ability to insert in or near to them. Four transposons, piggyBac, Mos1, Hermes and Minos are commonly used in insects beyond Drosophila melanogaster with piggyBac, due to its wide host range and frequency of transposition, being the most commonly chosen. The utility of these transposons as genetic tools is directly proportional to their activity since higher transposition rates would be expected to lead to higher transformation frequencies and higher frequencies of insertion throughout the genome. As a consequence there is an ongoing need for hyperactive transposases for use in insect genetics, however these have proven difficult to obtain. IPB7 is a hyperactive mutant of the piggyBac transposase that was identified by a genetic screen performed in yeast, a mammalian codon optimized version of which was then found to be highly active in rodent embryonic stem cells with no apparent deleterious effects. Here we report the activity of IPB7 in D. melanogaster and the mosquito, Aedes aegypti. Somatic transposition assays revealed an increase in IPB7's transposition rate from wild-type piggyBac transposase in D. melanogaster but not Ae. aegypti. However the use of IPB7 in D. melanogaster genetic transformations produced a high rate of sterility and a low transformation rate compared to wild-type transposase. This high rate of sterility was accompanied by significant gonadal atrophy that was also observed in the absence of the piggyBac vector transposon. We conclude that IPB7 has increased activity in the D. melanogaster germ-line but that a component of the sterility associated with its activity is independent of the presence of the piggyBac transposon.     

Inter-species sequence comparison of Brachypodium reveals how transposon activity corrodes genome colinearity

Intergenic sequences evolve rapidly in plant genomes through a process known as genomic turnover. To investigate the influence of DNA transposons on genomic turnover, we compared 1 Mbp of orthologous genomic sequences from Brachypodium distachyon and Brachypodium sylvaticum. We found that B. distachyon and B. sylvaticum diverged approximately 1.7-2.0 million years ago. Of a total of 219 genes identified on the analyzed sequences, 211 were colinear. However, only 24 transposable elements of a total of 451 were orthologous (i.e. inserted in the common ancestor). We characterized in detail 59 insertions and 60 excisions of DNA transposons in one or other species, which altered 17% of the intergenic space. The DNA transposon excision sites showed complex and highly diagnostic sequence motifs for double-strand break (DSB) repair. DNA transposon excisions can lead to extensive deletions of hundreds of base pairs of flanking sequence if the DSB is repaired by 'single-strand annealing', or insertions of up to several hundred base pairs of 'filler DNA' if the DSB is repaired by 'synthesis-dependent strand annealing'. In some cases, DSBs were repaired by a combination of both methods. We present a model for the evolution of intergenic sequences in which repair of DSBs upon DNA transposon excision is a major factor in the rapid turnover and erosion of intergenic sequences.     

<Emphasis Type="Italic">Mutator</Emphasis>-Based Transposon Display: A Genetic Tool for Evolutionary and Crop-Improvement Studies in Maize

Transposable elements account for up to 85% of the maize genome and have significant implications in crop-improvement and evolutionary analyses. The Mutator (Mu) transposon superfamily, a class of DNA transposons, comprises the most complex and active elements in the maize genome, suggesting a special role in plant evolution. Here, we designed a set of Mu-specific primers based on terminal invert repeats and used a transposon display (TD) method for genotyping. We analyzed the distribution pattern of Mu insertions in teosinte (wild relative), sorghum (distant relative), and domesticated maize accessions (dent, sweet, and waxy). The MU-TD analysis suggested the presence of high polymorphic insertions among the species and subspecies, indicating the utility of the method in studying genetic variation and species relationships. Furthermore, we analyzed 80 maize recombinant inbred line populations. Mu-TD generated an average of 60% Mu-anchored polymorphic fragments in which insertions appeared to be segregating in significantly high numbers. The amplification profile was highly reproducible, confirming the utility of Mu elements as a new set of TD markers for developing high-density genetic maps.     

Mu-seq: Sequence-Based Mapping and Identification of Transposon Induced Mutations

Mutations tagged by transposon insertions can be readily mapped and identified in organisms with sequenced genomes. Collections of such mutants allow a systematic analysis of gene function, and can be sequence-indexed to build invaluable resources. Here we present Mu-seq (Mutant-seq), a high-throughput NextGen sequencing method for harnessing high-copy transposons. We illustrate the efficacy of Mu-seq by applying it to the Robertson’s Mutator system in a large population of maize plants. A single Mu-seq library, for example, constructed from 576 different families (2304 plants), enabled 4, 723 novel, germinal, transposon insertions to be detected, identified, and mapped with single base-pair resolution. In addition to the specificity, efficiency, and reproducibility of Mu-seq, a key feature of this method is its adjustable scale that can accomodate simultaneous profiling of transposons in thousands of individuals. We also describe a Mu-seq bioinformatics framework tailored to high-throughput, genome-wide, and population-wide analysis of transposon insertions.     

Mos as a tool for genome-wide insertional mutagenesis in Caenorhabditis elegans: results of a pilot study

The sequence of the Caenorhabditis elegans genome contains approximately 19 000 genes. Available mutants currently exist for <20% of these genes. The existence of a Mos-based inducible transposon system in C.elegans could theoretically serve as a tool to saturate the genome with insertions. We report here the results of a pilot study aimed at assaying this strategy. We generated 914 independent random Mos insertions and determined their location by inverse PCR. The distribution of the insertions throughout the genome does not reveal any gross distortion, with the exception of a major hotspot on chromosome I (rDNA locus). Transposons are evenly distributed between the genic and intergenic regions. Within genes, transposons insert preferentially into the introns. We derived the consensus target site for Mos in C.elegans (ATATAT), which is common to Tc1, another mariner element. Finally, we assayed the mutagenic properties of insertions located in exons by comparing the phenotype of homozygous strains to that of known mutations or RNAi of the same gene. This pilot experiment shows that a Mos-based approach is a viable strategy that can contribute to the constitution of genome-wide collections of identified C.elegans mutants.     

Comparative Analysis of the Recently Discovered hAT Transposon TcBuster in Human Cells

Background

Transposons are useful tools for creating transgenic organisms, insertional mutagenesis, and genome engineering. TcBuster, a novel hAT-family transposon system derived from the red flour beetle Tribolium castaneum, was shown to be highly active in previous studies in insect embryoes.

Methodology/Principal Findings

We tested TcBuster for its activity in human embryonic kidney 293 (HEK-293) cells. Excision footprints obtained from HEK-293 cells contained small insertions and deletions consistent with a hAT-type repair mechanism of hairpin formation and non-homologous end-joining. Genome-wide analysis of 23,417 piggyBac, 30,303 Sleeping Beauty, and 27,985 TcBuster integrations in HEK-293 cells revealed a uniquely different integration pattern when compared to other transposon systems with regards to genomic elements. TcBuster experimental conditions were optimized to assay TcBuster activity in HEK-293 cells by colony assay selection for a neomycin-containing transposon. Increasing transposon plasmid increased the number of colonies, whereas gene transfer activity dependent on codon-optimized transposase plasmid peaked at 100 ng with decreased colonies at the highest doses of transposase DNA. Expression of the related human proteins Buster1, Buster3, and SCAND3 in HEK-293 cells did not result in genomic integration of the TcBuster transposon. TcBuster, Tol2, and piggyBac were compared directly at different ratios of transposon to transposase and found to be approximately comparable while having their own ratio preferences.

Conclusions/Significance

TcBuster was found to be highly active in mammalian HEK-293 cells and represents a promising tool for mammalian genome engineering.     

Target-site preferences of Sleeping Beauty transposons

The Sleeping Beauty (SB) transposon is a Tc1/mariner family transposon that has applications in vertebrate animals for gene transfer, gene-tagging, and human gene therapy. In this study, we analyzed the target-site preferences of the SB transposon. At the genomic level, integration of SB transposons with respect to genes (exons and introns) and intergenic regions appears fairly random but not on a micro-scale. Although there appears to be a consensus sequence around the vicinity of the target sites, the primary sequence is not the determining factor for target selection. When integrations were examined over a limited topography, the sites used most often for integration did not match the consensus sequence. Rather, a unique deformation inherent in the sequence may be a recognition signal for target selection. The deformation is characterized by an angling of the target site such that the axis around the insertion site is off-center, the rotation of the helix (twisting) is non-uniform and there is an increase in the distance between the central base-pairs. Our observations offer several hypothetical insights into the transposition process. Our observations suggest that particular deformations of the double helix predicted by the V(step) algorithm can distinguish TA sites that vary by about 16-fold in their preferences for accommodating insertions of SB transposons.     

Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.