57 research outputs found
Recombinase mediated cassette exchange into genomic targets using an adenovirus vector
Recombinase mediated cassette exchange (RMCE) is a process in which site-specific recombinases exchange one gene cassette flanked by a pair of incompatible target sites for another cassette flanked by an identical pair of sites. Typically one cassette is present in the host genome, whereas the other gene cassette is introduced into the host cell by chemical or biological means. We show here that the frequency of cassette exchange is dependent on the relative and absolute quantities of the transgene cassette and the recombinase. We were able to successfully modify genomic targets not only by electroporation or chemically mediated gene transfer but also by using an adenovirus vector carrying both the transgene cassette to be inserted and the recombinase coding region. RMCE proceeds efficiently in cells in which the adenovirus vector is able to replicate. In contrast, insufficient quantities of the transgene cassette are produced in cells in which the virus cannot replicate. Additional transfection of the transgene cassette significantly enhances the RMCE frequency. This demonstrates that an RMCE system in the context of a viral vector allows the site directed insertion of a transgene into a defined genomic site
Directed evolution of recombinase specificity by split gene reassembly
The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications
Zinc-finger recombinase activities in vitro
Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse ‘Z-sites’, which each comprise a central core sequence flanked by zinc-finger domain-binding motifs. Here, we show that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro. No off-site activity was detected. Under different reaction conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly dependent on the length of the Z-site core sequence. We show that this length effect is manifested at reaction steps prior to formation of recombinants (binding, synapsis and DNA cleavage). The design of the ZFR protein itself is also a crucial variable affecting activity. A ZFR with a very short (2 amino acids) peptide linkage between the catalytic and zinc-finger domains has high activity in vitro, whereas a ZFR with a very long linker was less recombination-proficient and less sensitive to variations in Z-site length. We discuss the causes of these phenomena, and their implications for practical applications of ZFRs
Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites
As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites
Selection of bacteriophage λ integrases with altered recombination specificity by in vitro compartmentalization
In vitro compartmentalization (IVC) was employed for the first time to select for novel bacteriophage λ integrase variants displaying significantly enhanced recombination activity on a non-cognate target DNA sequence. These variants displayed up to 9-fold increased recombination activity over the parental enzyme, and one mutant recombined the chosen non-cognate substrate more efficiently than the parental enzyme recombined the wild-type DNA substrate. The in vitro specificity phenotype extended to the intracellular recombination of episomal vectors in HEK293 cells. Surprisingly, mutations conferring the strongest phenotype do not occur in the λ integrase core-binding domain, which is known to interact directly with cognate target sequences. Instead, they locate to the N-terminal domain which allosterically modulates integrase activity, highlighting a previously unknown role for this domain in directing integrase specificity. The method we describe provides a robust, completely in vitro platform for the development of novel integrase reagent tools for in vitro DNA manipulation and other biotechnological applications
Regulated gene insertion by steroid-induced ΦC31 integrase
Nonviral integration systems are widely used genetic tools in transgenesis and play increasingly important roles in strategies for therapeutic gene transfer. Methods to efficiently regulate the activity of transposases and site-specific recombinases have important implications for their spatiotemporal regulation in live transgenic animals as well as for studies of their applicability as safe vectors for genetic therapy. In this report, strategies for posttranslational induction of a variety of gene-inserting proteins are investigated. An engineered hormone-binding domain, derived from the human progesterone receptor, hPR891, and specifically recognized by the synthetic steroid mifepristone, is fused to the Sleeping Beauty, Frog Prince, piggyBac and Tol2 transposases as well as to the Flp and ΦC31 recombinases. By analyzing mifepristone-directed inducibility of gene insertion in cultured human cells, efficient posttranslational regulation of the Flp recombinase and the ΦC31 integrase is documented. In addition, fusion of the ΦC31 integrase with the ERT2 modified estrogen receptor hormone-binding domain results in a protein, which is inducible by a factor of 22-fold and retains 75% of the activity of the wild-type protein. These inducible ΦC31 integrase systems are important new tools in transgenesis and in safety studies of the ΦC31 integrase for gene therapy applications
Recombinase technology: applications and possibilities
The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes
Directed evolution of a recombinase for improved genomic integration at a native human sequence
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