Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.
Nucleic Acids Res
; 41(19): 9197-207, 2013 Oct.
Article
em En
| MEDLINE
| ID: mdl-23921635
ABSTRACT
Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.
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Base de dados:
MEDLINE
Assunto principal:
Elementos de DNA Transponíveis
/
Marcação de Genes
/
Transposases
/
Proteínas de Ligação a DNA
Limite:
Humans
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article