Open AccessTranscription activator like effector (TALE)-directed piggyBac transposition in human cells
Author(s) -
Jesse B. Owens,
Damiano Mauro,
Ilko Stoytchev,
Mital S. Bhakta,
MoonSoo Kim,
David J. Segal,
Stefan Moisyadi
Publication year - 2013
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gkt677
Subject(s) - biology , insertional mutagenesis , transposase , transcription activator like effector nuclease , transposable element , genetics , effector , crispr , genome editing , insertion , transposition (logic) , gene , gene targeting , mutagenesis , homologous recombination , plasmid , dna , transgene , computational biology , genome , microbiology and biotechnology , mutation , linguistics , philosophy
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.