Transposons are natural gene delivery vehicles. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in the cells of vertebrates including humans. SB transposition into chromosomal DNA occurs in a fairly random manner. This is clearly not desirable in human gene therapeutic applications because there are potential genotoxic effects associated with transposon integration. In this study we set out to manipulate the selection of SB's target sites for targeted transposition into predetermined chromosomal regions. We evaluated experimental strategies based on engineered proteins composed of DNA-binding domains fused to (i) the transposase; (ii) another protein that binds to a specific DNA sequence within the transposable element; and (iii) another protein that interacts with the transposase. We demonstrated targeted transposition into endogenous matrix attachment regions (MARs) and a chromosomally integrated tetracycline response element (TRE) in cultured human cells, using targeting proteins that bind to the transposon DNA. An approach based on interactions between the transposase and a targeting protein containing the N-terminal protein interaction domain of SB was found to enable an approximately 10(7)-fold enrichment of transgene insertion at a desired locus. Our experiments provide proof-of-principle for targeted chromosomal transposition of an otherwise randomly integrating transposon. Targeted transposition can be a powerful technology for safe transgene integration in human therapeutic applications.
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http://dx.doi.org/10.1038/sj.mt.6300169 | DOI Listing |
J Clin Med
December 2024
Plastic Surgery Unit, University Hospital Trust of Sassari, 07100 Sassari, Italy.
Involutional lower eyelid ectropion is a common disorder of the elderly population. Several surgical approaches have been described in the literature to address the multifactorial nature of this condition, each targeting different factors contributing to its development. Nevertheless, no single procedure has proven to be superior to the others.
View Article and Find Full Text PDFMicroorganisms
December 2024
Department of Molecular Biology, School of Biological Sciences, University of California at San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0116, USA.
Insertions of the transposable element IS5 into its target sites in response to stressful environmental conditions, DNA structures, and DNA-binding proteins are well studied, but how the genomic contexts near IS5's native loci impact its transpositions is largely unknown. Here, by examining the roles of all 11 copies of IS5 within the genome of strain BW25113 in transposition, we reveal that the most significant copy of IS5 is one nested within and oriented in the same direction as the gene, while two other copies of IS5 harboring point mutations are hardly transposed. Transposition activity is heavily reliant on the upstream promoter that drives IS5 transposase gene , with more transpositions resulting from greater promoter activity.
View Article and Find Full Text PDFRadiol Imaging Cancer
January 2025
From the Department of Clinical Affairs, MediView XR, Cleveland, Ohio (M.E.); College of Medicine, Alfaisal University, Riyadh, Saudi Arabia (A.B.); and Department of Diagnostic Radiology, Section of Interventional Radiology, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195-5243 (S.K., K.G., C.M.).
Percutaneous tumor ablation has become a widely accepted and used treatment option for both soft and hard tissue malignancies. The current standard-of-care techniques for performing these minimally invasive procedures require providers to navigate a needle to their intended target using two-dimensional (2D) US or CT to obtain complete local response. These traditional image-guidance systems require operators to mentally transpose what is visualized on a 2D screen into the inherent three-dimensional (3D) context of human anatomy.
View Article and Find Full Text PDFAlgorithms Mol Biol
December 2024
Instituto de Computação, Universidade Federal Fluminense, Niterói, Brazil.
Genome rearrangements are events where large blocks of DNA exchange places during evolution. The analysis of these events is a promising tool for understanding evolutionary genomics, providing data for phylogenetic reconstruction based on genome rearrangement measures. Many pairwise rearrangement distances have been proposed, based on finding the minimum number of rearrangement events to transform one genome into the other, using some predefined operation.
View Article and Find Full Text PDFNucleic Acids Res
January 2025
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes.
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