Galli Alvaro, Cervelli Tiziana
Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via Moruzzi 1, 56125 Pisa, Italy.
BMC Mol Biol. 2014 Feb 13;15:5. doi: 10.1186/1471-2199-15-5.
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.
同源重组介导的基因打靶效率仍然过低，无法在基因组学和基因治疗中广泛应用。尽管序列特异性核酸酶可极大地提高基因打靶效率，但这些核酸酶的脱靶切割位点凸显了该策略的风险。基于腺相关病毒（AAV）的载体用于特定基因敲除，因为多项研究表明这些载体能够高频诱导位点特异性基因组改变。由于每个靶向事件至少伴随着十次随机整合事件，因此有必要增加我们对这些事件背后机制的了解，以便理解AAV介导的基因打靶在治疗应用中的潜力。此外，AAV调节蛋白（Rep）和反向末端重复序列（ITR）在随机整合和同源整合中的作用尚不完全清楚。在本研究中，我们使用酿酒酵母作为遗传模型系统，以评估整合质粒中ITR的存在是否对基因打靶和随机整合有影响。
我们已经表明，在与基因组靶点具有同源性的基因打靶载体两侧存在ITR会降低随机整合的频率，从而导致基因打靶/随机整合比率增加。另一方面，在ITR中产生切口的Rep蛋白的表达显著增加了与基因组无同源性的DNA片段的非同源整合，但当DNA片段与基因组具有同源性时，对基因打靶或随机整合没有影响。分子分析表明，ITR在随机整合体中经常保守，并且它们会诱导重排。
我们的结果表明，ITR可能是降低随机整合、从而有利于同源基因打靶的有用工具。
