Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes.

中文翻译：

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多合一，自靶向AAV载体在体内进行精确Cas9基因组编辑

腺相关病毒（AAV）载体是治疗基因组编辑的重要传递平台，但受到货物限制的严格限制，尤其是对于像Cas9s这样的大型效应子。同时递送多个载体会限制剂量和功效并增加安全风险。紧凑型效应器的使用已使带有1-3个指南的Cas9s的单AAV递送能够进行使用末端连接修复途径的编辑，但是许多纠正体内致病突变的精确编辑都需要同源直接修复（HDR）模板。在这里，我们描述了表达Nme2Cas9的两个单载体，〜4.8-kb AAV平台以及两个产生片段缺失的sgRNA或一个带有HDR模板的sgRNA。我们还检查了载体中Nme2Cas9目标位点对自我灭活的实用性。我们证明这些平台可以有效治疗小鼠中的两种疾病模型[I型遗传性酪氨酸血症（HT-I）和I型粘多糖贮积症（MPS-1）]。这些结果将使单载体AAV能够实现多种治疗性基因组编辑结果。