title: Retargeting transposon insertions by the adeno-associated virus Rep protein.
abstract: The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein-protein and protein-DNA interactions.
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Ismahen
authors.initials: I
authors.lastname: Ammar
authors.forename: Andreas
authors.initials: A
authors.lastname: Gogol-Döring
authors.forename: Csaba
authors.initials: C
authors.lastname: Miskey
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Izsvák
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Ivics
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019895:Transposases / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D016662:Virus Integration
chemical_list: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D004247:DNA; D019895:Transposases; C494471:sleeping beauty transposase, human