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Integration preferences of wildtype AAV-2 for consensus rep-binding sites at numerous loci in the human genome.

PLoS pathogens | 2010

Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.

Pubmed ID: 20628575 RIS Download

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NCBI Genome Survey Sequences Database (tool)

RRID:SCR_002146

Database of unannotated short single-read primarily genomic sequences from GenBank including random survey sequences clone-end sequences and exon-trapped sequences. The GSS division of GenBank is similar to the EST division, with the exception that most of the sequences are genomic in origin, rather than cDNA (mRNA). It should be noted that two classes (exon trapped products and gene trapped products) may be derived via a cDNA intermediate. Care should be taken when analyzing sequences from either of these classes, as a splicing event could have occurred and the sequence represented in the record may be interrupted when compared to genomic sequence. The GSS division contains (but is not limited to) the following types of data: * random single pass read genome survey sequences. * cosmid/BAC/YAC end sequences * exon trapped genomic sequences * Alu PCR sequences * transposon-tagged sequences Although dbGSS sequences are incorporated into the GSS Division of GenBank, annotation in dbGSS is more comprehensive and includes detailed information about the contributors, experimental conditions, and genetic map locations.

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RRID:SCR_001237

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RRID:SCR_005476

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RRID:SCR_011919

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