Inverted terminal repeats of adeno-associated virus decrease random integration of a gene targeting fragment in Saccharomyces cerevisiae
© Galli and Cervelli; licensee BioMed Central Ltd. 2014
Received: 16 October 2013
Accepted: 6 February 2014
Published: 13 February 2014
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.
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.
Our results indicate that ITRs may be a useful tool for decreasing random integration, and consequently favor homologous gene targeting.
KeywordsYeast AAV ITRs Homologous recombination Random integration
Gene targeting is the process by which exogenously delivered DNA is used to modify a genomic target by homologous recombination. Since in human cells the spontaneous frequency of homologous recombination is on the order of 10-6, the gene targeting approach is not efficient enough to be applied for clinical use . However, several reports indicate that sequence-specific nucleases, which induce a site-specific double strand break in the target DNA, can increase gene targeting efficiency up to 50,000-fold ; this approach is thus particularly useful for manipulating primary human cells with therapeutic potential [3, 4]. Although there has been considerable excitement about the potential application of zinc finger nucleases, since these enzymes allow highly specific, targeted genome modification in live cells , several reports show the off-target cleavage sites of these nucleases, highlighting the risk of using this approach [6, 7]. Therefore, the search for new tools to improve the efficiency of gene targeting is still very important. Recently, the induction of a site-specific single-strand nick significantly increased gene targeting . Our previously published studies show that the overexpression or nuclear permeation of the Saccharomyces cerevisiae protein Rad52 strongly increases homologous recombination and gene targeting in HeLa cells [9, 10]. An alternative method of homologous recombination mediated gene targeting consists of exploiting the recombinogenic nature of the adeno-associated virus (AAV) vector genome . The AAV single stranded (ss) DNA genome contains two overlapping open reading frames flanked by two inverted repeated sequences (ITRs), the only elements required in cis for replication and integration. The Rep open reading frame codes for Rep proteins essential for DNA replication, integration and packaging. The Cap codes for the proteins essential for capsid formation . Vectors based on AAV, which deliver single-stranded, linear DNA genomes, are able to efficiently introduce many types of mutations into homologous target loci at a frequency approaching 1% in mammalian cells, and are currently used as gene targeting vectors [13–16]. However, using this method, each homologous targeted event occurs within ten random integrations . Recently, by combining AAV technology with zinc finger nucleases, the efficiency of gene targeting increases up to 6% but most integration events still occur outside the target locus, most likely in naturally occurring DNA double-strand breaks [7, 15–18].
Moreover, AAV-mediated gene targeting has been reported to be less dependent on the extent of homology between the vector and the genome target than other methods . Notably, the presence of the ITRs flanking the gene targeting construct determines increased homologous recombination frequencies .
When AAV Rep proteins are expressed, the yeast Saccharomyces cerevisiae is able to replicate the ssDNA genome . The proteins Rep68 and Rep40 are necessary for AAV replication and for site-specific integration [21, 22]. Rep proteins interact with the Rep-binding element and the terminal resolution site sequences located within the ITRs, to create a nick that may increase the integration [23, 24].
In the yeast Saccharomyces cerevisiae, gene targeting is very efficient; it occurs at a frequency of 2–3% and can be increased up to 25%, whereas random or non-homologous integration is reported to be less than 0.1% [25, 26]. Yeast is an excellent genetic model for understanding the mechanisms and pathways involved in homologous and non-homologous recombination [27–30]. Moreover, yeast has been recently used as a system for studying the potential of a new genome editing approach for site-specific mutagenic and multiple allele replacement .
We decided to use the yeast Saccharomyces cerevisiae to evaluate whether the presence of ITRs in the integrating plasmid and the expression of AAV Rep proteins have an effect on gene targeting and random integration. The aim of this study was to assess whether the use of AAV sequences or expression of Rep proteins could be a feasible and valuable tool for increasing gene targeting or decreasing random integration.
Results and discussion
The presence of ITRs decreased the random integration of a gene targeting construct
Effect of the AAV ITR sequence on gene targeting and random integration in yeast
31.0 ± 11.3 (7025)
0.62 ± 0.37 (487)
35.4 ± 10.4 (6960)
1.82 ± 0.41 (3420)*
The expression of Rep proteins increased AAV integration of a non-homologous fragment carrying ITRs
Effect of the AAV Rep protein expression on gene targeting and random integration in yeast
ITR-lys2URA3lys2-ITR, No REP
31.8 ± 9.8
0.73 ± 0.39
20.1 ± 5.9
0.88 ± 0.49
ITR-pokURA3-ITR, No REP
0.12 ± 0.08
1.02 ± 0.56*
Our results indicate that the ITRs and Rep proteins may affect AAV genomic random integration when no homology between the vector and the genome is present, and that rearrangements occur. Conversely, the presence of the AAV ITRs at both ends in a gene targeting construct sharing homology with a genomic locus could have an impact on the application of this strategy by significantly decreasing random integration.
The construction of the plasmid pAAVPokURA, which contains the URA3 marker and the 2.5 kb Pok stuffer sequence to increase the distance between the ITRs, has been previously reported  (Figure 1A). The plasmid pAAVLUL carrying the gene-targeting fragment between the two ITRs (Figure 1B), was constructed as follows: the 2.7 kb Eco RI-Bam HI fragment from pJZ102  was first cloned into the pMCSsub; then, the entire fragment was cut off from pMCSsub with Xba I and inserted directly into the Xba I site of pSub201 containing the ITRs .
Yeast transformation and molecular analysis
The yeast strain RSY12 (MATa leu2–3,112 his3–11,15 URA3::HIS3) has a complete deletion of the URA3 gene, which was replaced with the HIS3 gene . To evaluate the effect of Rep proteins expression on gene targeting and random integration, we transformed the pAAV vectors in the RSY12 yeast strain containing the vector pG.Rep68 integrated into the genome .
Complete (YPAD) and synthetic complete (SC) medium were prepared according to standard procedures. Yeast was transformed with 3–5 μg of plasmid DNA using the standard lithium chloride method with single-stranded DNA as carrier . The vectors pAAVLUL and pAAVpokURA were transformed, digested with either Pvu II or Xba I. Transformants were selected on SC-uracil (SC-URA). As the gene targeting events disrupt the chromosomal LYS2 gene by inserting the URA3 marker, the URA3 transformants were replica-plated in SC-lysine-uracil (SC-LYS -URA) plates to score for the random integration and gene targeting. In parallel, the same yeast culture was transformed with an episomal plasmid to evaluate the efficiency of transformation per μg of plasmid DNA. The frequency of gene targeting and random integration, calculated by dividing the number of events by the transformation efficiency, was expressed as number of URA+lys2-/103 and URA+LYS2+/103 total transformants per μg of plasmid DNA, respectively. Data were statistically evaluated by Student’s t-test with computer assistance. Single clones were grown in 5–10 ml of SC-URA and the genomic DNA isolated using the Master pure yeast DNA purification Kit (Epicentre Biotechnologies). The DNA was digested with Ase I, which does not cut in the rAAV fragment, electrophoresed, transferred to a nylon membrane (Roche), and hybridized with DIG-labelled -URA3 and ITR probes according to the standard protocol. DIG-labelled URA3 and ITR were obtained as previously described .
Western blot was performed with the monoclonal antibody 303.9 (PROGEN, Germany) and anti-3PGK (Invitrogen) as previously described .
To clone the junction sites, the genomic DNA from URA3+LYS2+ clones was digested with Sph I and ligated in the Sph I site of YEplac181 . Then, the ligation mixture was transformed into competent E.coli. Single ampicillin-resistant colonies were grown in selection liquid medium, plasmid DNA extracted and sequenced with these primers: LYS2-UP 5’ TCCACTGCCAAGTATAGAA 3’ and LYS2-LOW 5’GTCATGTGGTAACACTGAA 3’.
The authors are grateful to Robert Schiestl for the RSY12 yeast strain and the pJZ102 plasmid. The authors wish to thank Ana Backovic and Lidia Balestri for technical assistance and helpful suggestions. They are grateful to Marcella Simili for reading and editing the final version of the manuscript.
This work was supported by Grant n. GGP09166 assigned by Telethon-Italy to AG. The funder had no role in study design, data collection, decision to publish or preparation of the manuscript.
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