Transient expression of the CRISPR-Cas9 machinery is desirable to reduce the risks of off-targets and immune responses. Electroporation of Cas9 ribonucleoproteins (RNPs) is the most common delivery method to achieve transient Cas9 expression. Recently, retroviral capsids have been used for delivering Streptococcus pyogenes Cas9 RNPs, in which Cas9 was fused to the viral proteins. The fusion strategy may cause relative low capsid assembly efficiency. We recently developed virus-like particles (VLPs) consisting of lentiviral capsid and Staphylococcus aureus Cas9 RNPs using the specific interactions between aptamer and aptamer-binding protein (ABP), and obtained near-normal capsid assembly efficiency. Here we test whether highly active Streptococcus pyogenes Cas9 (SpCas9) RNP VLPs can be generated with high efficiency by aptamer/ABP interaction. We found that by optimizing the locations and types of aptamer used for single guide RNA modification, highly active SpCas9 RNP VLPs can be generated efficiently. VLP-delivered SpCas9 generated lower off-target insertions and deletions than SpCas9 RNPs delivered by electroporation. VLPs containing Cas9 from different species and targeting multiple genes can be efficiently prepared in single-particle preparation. Multiple-target VLPs were more efficient than the combination of single-target VLPs for simultaneous targeting of multiple genes. Thus, in addition to better safety features, the Cas9 VLPs are especially suited for multiplex genome editing. In summary, our VLPs offer safe, efficient, and flexible multiplex genome editing.

中文翻译：

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慢病毒衣壳介导的化脓性链球菌 Cas9 核糖核蛋白递送用于高效和安全的多重基因组编辑

CRISPR-Cas9 机器的瞬时表达对于降低脱靶和免疫反应的风险是可取的。Cas9 核糖核蛋白 (RNP) 的电穿孔是实现瞬时 Cas9 表达的最常见的递送方法。最近，逆转录病毒衣壳已被用于传递化脓性链球菌Cas9 RNP，其中 Cas9 与病毒蛋白融合。融合策略可能导致衣壳组装效率相对较低。我们最近利用适体和适体结合蛋白 (ABP) 之间的特异性相互作用开发了由慢病毒衣壳和金黄色葡萄球菌Cas9 RNPs组成的病毒样颗粒 (VLPs) ，并获得了接近正常的衣壳组装效率。在这里我们测试是否是高度活跃的化脓性链球菌Cas9 (SpCas9) RNP VLPs可以通过适配体/ABP相互作用高效生成。我们发现，通过优化用于单向导 RNA 修饰的适体的位置和类型，可以有效地生成高活性 SpCas9 RNP VLP。与通过电穿孔传递的 SpCas9 RNP 相比，VLP 传递的 SpCas9 产生的脱靶插入和缺失更少。含有来自不同物种的 Cas9 并靶向多个基因的 VLP 可以在单颗粒制备中有效制备。对于同时靶向多个基因，多靶点 VLP 比单靶点 VLP 组合更有效。因此，除了更好的安全特性外，Cas9 VLP 特别适用于多重基因组编辑。总之，我们的 VLP 提供安全、高效和灵活的多重基因组编辑。