Inverted terminal repeats of adeno-associated virus decrease random integration of a gene targeting fragment in Saccharomyces cerevisiae

Abstract

Background: Homologous recombination mediated gene targeting is still too inefficient to be applied extensively in genomics and gene therapy. Although sequence-specific nucleases could greatly stimulate gene targeting efficiency, the off-target cleavage sites of these nucleases highlighted the risk of this strategy. Adeno-associated virus (AAV)-based vectors are used for specific gene knockouts, since several studies indicate that these vectors are able to induce site-specific genome alterations at high frequency. Since each targeted event is accompanied by at least ten random integration events, increasing our knowledge regarding the mechanisms behind these events is necessary in order to understand the potential of AAV-mediated gene targeting for therapy application. Moreover, the role of AAV regulatory proteins (Rep) and inverted terminal repeated sequences (ITRs) in random and homologous integration is not completely known. In this study, we used the yeast Saccharomyces cerevisiae as a genetic model system to evaluate whether the presence of ITRs in the integrating plasmid has an effect on gene targeting and random integration.

Results: We have shown that the presence of ITRs flanking a gene targeting vector containing homology to its genomic target decreased the frequency of random integration, leading to an increase in the gene targeting/random integration ratio. On the other hand, the expression of Rep proteins, which produce a nick in the ITR, significantly increased non-homologous integration of a DNA fragment sharing no homology to the genome, but had no effect on gene targeting or random integration when the DNA fragment shared homology with the genome. Molecular analysis showed that ITRs are frequently conserved in the random integrants, and that they induce rearrangements.

Conclusions: Our results indicate that ITRs may be a useful tool for decreasing random integration, and consequently favor homologous gene targeting.

Figure 1

Schematic representations of plasmids carrying the recombinant AAV fragment (rAAV). A) pAAVpokURA. B) pAAVLUL. In both plasmids, restriction with PvuII gives rise to the rAAV fragment containing the ITRs; restriction with XbaI cuts out the ITRs and generates a fragment with no ITR at both ends.

Figure 2

Molecular analysis of random integration clones derived from transformation with ITRs carrying fragment. Southern blot analysis of genomic DNA isolated from URA3⁺LYS2⁺ yeast transformant clones. These clones were obtained by transforming RSY12 yeast strain with the ITRs-containing fragment obtained by the digestion of pAAVLUL vector with PvuII. We analyzed genomic DNA digested with AseI of eleven different clones. The numbers above the filters indicate the clones. Bands were detected using the URA3 probe and the ITR probe as indicated.

Figure 3

Molecular analysis of random integration clones derived from transformation with no-ITRs-containing fragment. Southern blot analysis of genomic DNA from fourteen URA3⁺LYS2⁺ yeast clones derived from transformation of RSY12 yeast strain with pAAVLUL digested with XbaI. Digestion with XbaI produces a fragment without ITR as described in Figure 1B. Genomic DNA was digested with AseI that does not cut in the sequence between the ITRs. The numbers above the filters indicate the clones. Bands were detected using the URA3 probe.

Figure 4

Expression of Rep proteins and random integration of an AAV vector without homology with the yeast genome. A) Western blot analysis of total cell lysate from yeast cells not expressing (lane 1) and expressing Rep proteins (lane 2) and transformed with pAAVPokURA carrying the ITRs. PGK3 antibody is used as loading control. B) Southern blot of genomic DNA of clones derived from transformation of Rep expressing yeast strain with pAAVPokURA. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23: genomic DNA not restricted with AseI; lane 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24: genomic DNA digested with AseI that does not cut in the sequence between the ITRs containing Pok and URA3.