Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.

中文翻译：

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在体外和体内将单特异性和双特异性 Cas9/向导 RNA 递送至扰动基因的平台


尽管 CRISPR-Cas9 技术有望彻底改变具有潜在基因突变的疾病的治疗，但它面临着一些限制临床进入的重大问题。它们包括低效 体内系统递送和不希望的脱靶效应。在这里，我们证明，通过用硫代磷酸酯 DNA 寡核苷酸 （PS） 修饰 Cas9，可以在体外和 体内有效地递送单特异性和双特异性 CRISPR-Cas9/向导 RNA （gRNA） 二聚体 ，同时减少脱靶效应。我们表明 PS-Cas9/gRNA 介导的基因敲除在体外和 体内保留了嵌合抗原受体 T 细胞的活力和扩增 。PS-Cas9/gRNA 介导患者来源的肿瘤类器官和小鼠异种移植肿瘤中的基因扰动，从而产生有效的肿瘤抗肿瘤作用。此外，HER2 抗体-PS-Cas9/gRNA 偶联物在体内选择性扰乱 HER2 + 卵巢癌异种移植物 中的靶基因。此外，我们创造了具有两个 gRNA 的双特异性 PS-Cas9 来靶向同一基因的两个相邻序列，从而在体外 和 体内实现有效的靶向基因破坏，并显着减少了意外的基因扰动。因此，穿透细胞的 PS-Cas9/gRNA 可以实现有效的全身递送和基因破坏的精确性。