Precise targeted integration by a chimaeric transposase zinc-finger fusion protein
TLDR
A TA dinucleotide flanked by two Zif268 binding sites was efficiently targeted by the transposase-Zif268 fusion, suggesting the possibility of designer ‘Z-transposases’ that could deliver transgenic cargoes to chosen genomic locations.Abstract:
Transposons of the Tc1/mariner family have been used to integrate foreign DNA stably into the genome of a large variety of different cell types and organisms. Integration is at TA dinucleotides located essentially at random throughout the genome, potentially leading to insertional mutagenesis, inappropriate activation of nearby genes, or poor expression of the transgene. Here, we show that fusion of the zinc-finger DNA-binding domain of Zif268 to the C-terminus of ISY100 transposase leads to highly specific integration into TA dinucleotides positioned 6-17 bp to one side of a Zif268 binding site. We show that the specificity of targeting can be changed using Zif268 variants that bind to sequences from the HIV-1 promoter, and demonstrate a bacterial genetic screen that can be used to select for increased levels of targeted transposition. A TA dinucleotide flanked by two Zif268 binding sites was efficiently targeted by our transposase-Zif268 fusion, suggesting the possibility of designer ‘Z-transposases’ that could deliver transgenic cargoes to chosen genomic locations.read more
Citations
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Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy.
George H. Silva,Laurent Poirot,Roman Galetto,Julianne Smith,Guillermo Montoya,Philippe Duchateau,Frédéric Paques +6 more
TL;DR: These alternative approaches based on non-viral vectorization and/or targeted insertion aimed at achieving safer gene transfer are reviewed, with a special emphasis on megan nucleases, a family of naturally occurring rare-cutting endonucleases, and speculate on their current and future potential.
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Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology
Salvatore J. Orlando,Yolanda Santiago,Russell Dekelver,Yevgeniy Freyvert,Elizabeth A. Boydston,Erica A. Moehle,Vivian M. Choi,Sunita Gopalan,Jacqueline F. Lou,James Li,Jeffrey C. Miller,Michael C. Holmes,Philip D. Gregory,Fyodor D. Urnov,Gregory J. Cost +14 more
TL;DR: It is demonstrated that easily-generated linear donors with extremely short homology regions drive transgene integration into 5–10% of chromosomes and that oligonucleotide donors with single-stranded 5′ overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB.
Journal ArticleDOI
Everyman's Guide to Bacterial Insertion Sequences
TL;DR: This review presents an overview of the current understanding of these transposable elements (TE), their organization and their transposition mechanism as well as their distribution and genomic impact, and provides a detailed description of the expanding variety of IS.
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Insect Transgenesis: Current Applications and Future Prospects
TL;DR: Limitations of transposon systems were identified, and alternative tools were developed, thus significantly increasing the potential for applied transgenics for control of both agricultural and medical insect pests.
Patent
Nucleotide-specific recognition sequences for designer tal effectors
Feng Zhang,Le Cong +1 more
TL;DR: In this article, a deoxyribonucleic acid (DNA) binding polypeptide is encoded by and translated from a codon optimized nucleic acid molecule so that the polypeptin preferentially binds to the DNA of the genomic locus.
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