Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone 3′ end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

中文翻译：

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AAV 介导的基因组编辑受 R 环形成的影响


重组腺相关病毒载体 （rAAV） 具有刺激同源重组 （AAV-HR） 的内在能力，是临床上最常用的体内 基因治疗载体。然而，rAAV 也整合在整个基因组中。在这里，我们描述了小鼠 HEPA1-6 肝癌细胞和整个小鼠肝脏中的 DNA-RNA 免疫沉淀测序 （DRIP-seq），以确定转化细胞系和完整组织中基因组 R 环形成的相似性和差异性。我们显示 R 环的遗传和药理学上调后小鼠的 AAV-HR 增强。在体外和 体内实验中选择 高表达的白蛋白基因作为基因组编辑的模型位点表明，白蛋白的 R 环易发 3' 端被 AAV-HR 有效编辑，而上游 R 环缺陷区域不会导致可检测的载体整合。此外，我们发现先前报道的脱靶 rAAV 整合位点与 R 环富集基因组区域之间存在正相关关系。因此，我们得出结论，存在于高度转录基因中的高水平 R 环可能会促进 rAAV 载体基因组整合。这些发现可能阐明通过调节 R 环来提高基因组编辑安全性和有效性的潜在机制，并可能增强我们预测最容易受到 rAAV 载体脱靶插入诱变的区域的能力。