Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Language
Publication year range
1.
Nucleic Acids Res ; 50(5): 2807-2825, 2022 03 21.
Article in English | MEDLINE | ID: mdl-35188569

ABSTRACT

The Sleeping Beauty (SB) transposon system is a popular tool for genome engineering, but random integration into the genome carries a certain genotoxic risk in therapeutic applications. Here we investigate the role of amino acids H187, P247 and K248 in target site selection of the SB transposase. Structural modeling implicates these three amino acids located in positions analogous to amino acids with established functions in target site selection in retroviral integrases and transposases. Saturation mutagenesis of these residues in the SB transposase yielded variants with altered target site selection properties. Transposon integration profiling of several mutants reveals increased specificity of integrations into palindromic AT repeat target sequences in genomic regions characterized by high DNA bendability. The H187V and K248R mutants redirect integrations away from exons, transcriptional regulatory elements and nucleosomal DNA in the human genome, suggesting enhanced safety and thus utility of these SB variants in gene therapy applications.


Subject(s)
Transposases , Amino Acids/genetics , DNA Transposable Elements/genetics , Humans , Integrases/metabolism , Protein Engineering , Transposases/genetics , Transposases/metabolism
2.
Nucleic Acids Res ; 48(1): 316-331, 2020 01 10.
Article in English | MEDLINE | ID: mdl-31777924

ABSTRACT

The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int- transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int- transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change.


Subject(s)
Cellular Reprogramming , DNA Transposable Elements , Induced Pluripotent Stem Cells/metabolism , Transposases/genetics , Amino Acid Substitution , Animals , Epistasis, Genetic , Genetic Engineering/methods , HeLa Cells , Hep G2 Cells , Humans , Induced Pluripotent Stem Cells/cytology , Mice , Mutation , Transposases/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...