AAV Recombineering with Single Strand Oligonucleotides
Figure 7
A Model for Oligo-Assisted AAV Genome Recombination.
Following the capsid release of the 4.7 kb AAV genomes, presumably in the nucleus, oligos with homology to each vector anneal to their single-strand (ss) target, displacing the remaining terminal ends of the genome. This annealing event promotes the desired genome dimer orientation and viral genome replication using the inverted terminal repeats for initiation. Extension of the 3′ oligo end is also a possibility. Exploitation of host repair enzymes for intermolecular recombination follows resulting in a double strand product with the vector junction sequence corresponding to that of the oligo. In the case of the H-Ib oligo this recombination event results in an approximate 400 bp deletion of the ITR junction. The recombined dimer is near double the length of the independent genomes. Although it was not conclusively demonstrated, this model favors physical incorporation of the transfected oligo into the oligo-directed concatemer (described in discussion). Also depicted are perhaps the naturally favored products initially following transduction, circular monomers, which do not produce GFP.