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 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 and with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion and . 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 . 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 and 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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一个提供单特异性和双特异性 Cas9/引导 RNA 来扰乱体外和体内基因的平台


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。它们包括低效率的全身递送和不良的脱靶效应。在这里，我们证明，通过用硫代磷酸酯-DNA 寡核苷酸 (PS) 修饰 Cas9，可以有效地递送单特异性和双特异性 CRISPR-Cas9/引导 RNA (gRNA) 二聚体，并减少脱靶效应。我们证明 PS-Cas9/gRNA 介导的基因敲除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 缀合物选择性干扰 HER2 卵巢癌异种移植物中的靶基因。此外，我们创建了具有两个 gRNA 的双特异性 PS-Cas9，以靶向同一基因的两个相邻序列，从而实现有效的靶向基因破坏，并显着减少意外的基因扰动。因此，细胞穿透型 PS-Cas9/gRNA 可以实现高效的系统递送和基因破坏的精确性。