We previously developed an adeno-associated virus (AAV) Cas9 gene therapy for Angelman syndrome that integrated into the genome and prematurely terminated Ube3a-ATS. Here, we assessed the performance of 3 additional AAV vectors containing S. aureus Cas9 in vitro and in vivo, and 25 vectors containing N. meningitidis Cas9 in vitro, all targeting single sites within Ube3a-ATS. We found that none of these single-target gRNA vectors were as effective as multi-target gRNA vectors at reducing Ube3a-ATS expression in neurons. We also developed an anchored multiplex PCR sequencing method and analysis pipeline to quantify the relative frequency of all possible editing events at target sites, including AAV integration and unresolved double-strand breaks. We found that integration of AAV was the most frequent editing event (67%–89% of all edits) at three different single target sites, surpassing insertions and deletions (indels). None of the most frequently observed indels were capable of blocking transcription when incorporated into a Ube3a-ATS minigene reporter, whereas two vector derived elements—the poly(A) and reverse promoter—reduced downstream transcription by up to 50%. Our findings suggest that the probability that a gene trapping AAV integration event occurs is influenced by which vector-derived element(s) are integrated and by the number of target sites.

中文翻译：

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AAV 载体衍生元件整合到 Cas9 产生的双链断裂中并破坏基因转录


我们之前开发了一种针对 Angelman 综合征的腺相关病毒 （AAV） Cas9 基因疗法，该疗法整合到基因组中并提前终止了 Ube3a-ATS。在这里，我们在体外 和体内评估了 3 个含有金黄色葡萄球菌 Cas9 的 AAV 载体，以及 25 个含有脑膜炎奈瑟菌 Cas9 的体外载体的性能，均靶向 Ube3a-ATS 内的单个位点。我们发现，这些单靶标 gRNA 载体在降低神经元中 Ube3a-ATS 表达方面均不如多靶标 gRNA 载体有效。我们还开发了一种锚定多重 PCR 测序方法和分析管道，以量化靶位点所有可能的编辑事件的相对频率，包括 AAV 整合和未解析的双链断裂。我们发现，AAV 整合是三个不同的单一靶位点最常见的编辑事件（占所有编辑的 67%-89%），超过了插入和删除 （插入缺失）。当掺入 Ube3a-ATS 小基因报告基因中时，最常观察到的插入缺失都无法阻断转录，而两个载体衍生元件（poly（A） 和反向启动子）将下游转录减少了高达 50%。我们的研究结果表明，基因捕获 AAV 整合事件发生的概率受整合的载体衍生元件和靶位点数量的影响。