The capacity to efficiently deliver the gene-editing enzyme complex to target cells is favored over other forms of gene delivery as it offers one-time hit-and-run gene editing, thus improving precision and safety and reducing potential immunogenicity against edited cells in clinical applications. Here we performed a proof-of-mechanism study and demonstrated that a simian adenoviral vector for DNA delivery can be repurposed as a robust intracellular delivery platform for a functional Cas9/guide RNA (gRNA) complex to recipient cells. In this system, the clinically relevant adenovirus was genetically engineered with a plug-and-display technology based on SpyTag003/SpyCatcher003 coupling chemistry. Under physiological conditions, an off-the-shelf mixture of viral vector with SpyTag003 incorporated into surface capsid proteins and Cas9 fused with SpyCatcher003 led to a rapid titration reaction yielding adenovirus carrying Cas9SpyCatcher003 on the virus surface. The Cas9 fusion protein-conjugated viruses in the presence of a reporter gRNA delivered gene-editing functions to cells with an efficiency comparable to that of a commercial CRISPR/Cas9 transfection reagent. Our data fully validate the adenoviral “piggyback” approach to deliver an intracellularly acting enzyme cargo and, thus, warrant the prospect of engineering tissue-targeted adenovirus carrying Cas9/gRNA for in vivo gene editing.

中文翻译：

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通过即插即用腺病毒在病毒衣壳表面搭载 Cas9/gRNA 复合物实现高效基因组编辑


与其他形式的基因递送相比，将基因编辑酶复合物有效递送至靶细胞的能力更受青睐，因为它提供了一次性基因编辑，从而提高了精度和安全性，并减少了临床中针对编辑细胞的潜在免疫原性。应用程序。在这里，我们进行了机制验证研究，并证明用于 DNA 递送的猿腺病毒载体可以重新用作强大的细胞内递送平台，用于将功能性 Cas9/引导 RNA (gRNA) 复合物递送至受体细胞。在该系统中，临床相关的腺病毒采用基于 SpyTag003/SpyCatcher003 耦合化学的即插即用技术进行基因工程改造。在生理条件下，现成的病毒载体与掺入表面衣壳蛋白的SpyTag003以及与SpyCatcher003融合的Cas9的现成混合物导致快速滴定反应，产生在病毒表面携带Cas9SpyCatcher003的腺病毒。 Cas9融合蛋白缀合病毒在报告基因gRNA存在的情况下向细胞传递基因编辑功能，其效率与商业CRISPR/Cas9转染试剂相当。我们的数据充分验证了腺病毒“搭载”方法来传递细胞内作用的酶货物，因此，保证了工程化携带Cas9/gRNA的组织靶向腺病毒用于体内基因编辑的前景。