Transcription activator like effector (TALE)-directed piggyBac transposition in human cells

Jesse B. Owens, Damiano Mauro, Ilko Stoytchev, Mital S. Bhakta, Moon Soo Kim, David Segal, Stefan Moisyadi

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)9197-9207
Number of pages11
JournalNucleic Acids Research
Volume41
Issue number19
DOIs
StatePublished - Oct 2013

ASJC Scopus subject areas

  • Genetics

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