119 research outputs found
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
A Genome-Wide Analysis of FRT-Like Sequences in the Human Genome
Efficient and precise genome manipulations can be achieved by the
Flp/FRT system of site-specific DNA recombination.
Applications of this system are limited, however, to cases when target sites for
Flp recombinase, FRT sites, are pre-introduced into a genome
locale of interest. To expand use of the Flp/FRT system in
genome engineering, variants of Flp recombinase can be evolved to recognize
pre-existing genomic sequences that resemble FRT and thus can
serve as recombination sites. To understand the distribution and sequence
properties of genomic FRT-like sites, we performed a
genome-wide analysis of FRT-like sites in the human genome
using the experimentally-derived parameters. Out of 642,151 identified
FRT-like sequences, 581,157 sequences were unique and
12,452 sequences had at least one exact duplicate. Duplicated
FRT-like sequences are located mostly within LINE1, but
also within LTRs of endogenous retroviruses, Alu repeats and other repetitive
DNA sequences. The unique FRT-like sequences were classified
based on the number of matches to FRT within the first four
proximal bases pairs of the Flp binding elements of FRT and the
nature of mismatched base pairs in the same region. The data obtained will be
useful for the emerging field of genome engineering
Cisgenesis and intragenesis as new strategies for crop improvement
Cisgenesis and intragenesis are emerging plant breeding technologies which offer great promise for future acceptance of genetically engineered crops. The techniques employ traditional genetic engineering methods but are confined to transferring of genes and genetic elements between sexually compatible species that can breed naturally. One of the main requirements is the absence of selectable marker genes (such as antibiotic resistance genes) in the genome. Hence the sensitive issues with regard to transfer of foreign genes and antibiotic resistance are overcome. It is a targeted technique involving specific locus; therefore, linkage drag that prolongs the time for crop improvement in traditional breeding does not occur. It has great potential for crop improvement using superior alleles that exist in the untapped germplasm or wild species. Cisgenic and intragenic plants may not face the same stringent regulatory assessment for field release as transgenic plants which is a clear added advantage that would save time. In this chapter, the concepts of cis/intragenesis and the prerequisites for the development of cis/intragenesis plants are elaborated. Strategies for marker gene removal after selection of transformants are discussed based on the few recent reports from various plant species
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
Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion
Isolating the Arabidopsis thaliana Genes for de Novo Purine Synthesis by Suppression of Escherichia coli Mutants (I. 5[prime]-Phosphoribosyl-5-Aminoimidazole Synthetase)
Use of Synthetic Hybrid Strains To Determine the Role of Replicon 3 in Virulence of the Burkholderia cepacia Complex
Conditional mutagenesis using site-specific recombination in Plasmodium berghei
Reverse genetics in Plasmodium, the genus of parasites that cause malaria, still faces major limitations. Only red blood cell stages of this haploid parasite can be transfected. Consequently, the function of many essential genes in these and subsequent stages, including those encoding vaccine candidates, cannot be addressed genetically. Here, we establish conditional mutagenesis in Plasmodium by using site-specific recombination and the Flp/FRT system of yeast. Site-specific recombination is induced after cross-fertilization in the mosquito vector of two clones containing either the target sequence flanked by two FRT sites or the Flp recombinase. Parasites that have undergone recombination are recognized in the cross progeny through the expression of a fluorescence marker. This approach should permit to dissect the function of any essential gene of Plasmodium during the haploid phase of its life, i.e., during infection of salivary glands in the mosquito and infection of both the liver and red blood cells in the mammal
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