The application of gene-editing technology is currently limited by the lack of safe and efficient methods to deliver RNA-guided endonucleases to target cells. We engineered lentivirus-based nanoparticles to co-package the U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. Equal spatiotemporal control of the vpr.prot.cas9 and gag/pol gene expression (the presence of Rev responsive element, RRE) greatly enhanced the encapsidation of the fusion protein and resulted in the production of highly efficient lentivector nanoparticles. Transduction of the unconcentrated, Vpr.Prot.Cas9-containing vectors led to >98% disruption of the EGFP gene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, we detected indels in the targeted endogenous loci at frequencies of up to 100% in cell lines derived from lymphocytes and monocytes and up to 15% in primary CD4+ T cells by high-throughput sequencing. This approach may provide a platform for the efficient, dose-controlled and tissue-specific delivery of genome editing enzymes to cells and it may be suitable for simultaneous endogenous gene disruption and a transgene delivery.

中文翻译：

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使用未浓缩的慢病毒载体进行高效的“即插即用”基因组编辑，该慢病毒载体携带由含有RRE的转录本产生的Vpr.Prot.Cas9蛋白。

目前，由于缺乏将RNA引导的核酸内切酶递送至靶细胞的安全有效方法，基因编辑技术的应用受到了限制。我们设计了基于慢病毒的纳米颗粒，将U6-sgRNA模板和CRISPR相关蛋白9（Cas9）与以病毒体为靶标的蛋白Vpr（Vpr.Prot.Cas9）融合在一起，以同时递送至细胞。vpr.prot.cas9和gag / pol基因表达的相同时空控制（Rev响应元件，RRE的存在）大大增强了融合蛋白的衣壳化，并产生了高效的慢病毒载体纳米颗粒。未浓缩的含有Vpr.Prot.Cas9的载体的转导导致EGFP的> 98％破坏该基因在报道的HEK293-EGFP细胞中具有最小的细胞毒性。此外，通过高通量测序，我们从淋巴细胞和单核细胞衍生的细胞系中以高达100％的频率检测到目标内源性基因座中的indel，在原代CD4 + T细胞中以15％的频率检测到了indel。该方法可以为基因组编辑酶向细胞的有效，剂量控制和组织特异性递送提供平台，并且它可能适合于同时进行内源基因破坏和转基因递送。