Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.
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.
Institute for Biogenesis Research, Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96822, USA, Genome Center, Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA and Manoa BioSciences, Honolulu, HI 96819, USA.
Jesse B
JB
Owens
Damiano
D
Mauro
Ilko
I
Stoytchev
Mital S
MS
Bhakta
Moon-Soo
MS
Kim
David J
DJ
Segal
Stefan
S
Moisyadi
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting; D057809:HEK293 Cells; D006801:Humans; D019713:Receptors, CCR5 / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
C584933:CCR5 protein, human; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; D019895:Transposases