Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome editing in mouse brain
Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome editing in mouse brain
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DOI：
10.1016/j.jconrel.2021.02.026
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发表时间：
2021-03-04
影响因子：
10.8
通讯作者：
Kataoka, Kazunori
中科院分区：
文献类型：
--
作者：
Abbasi, Saed;Uchida, Satoshi;Kataoka, Kazunori
使用 CRISPR/Cas9 进行基因组编辑在遗传性疾病和病毒感染的治疗方面引起了广泛关注。 Cas9 mRNA 和单引导 (sg)RNA 的共同传递是一种很有前途的策略，可以有效编辑各种细胞类型（包括非分裂细胞）的基因组，并且安全性问题最小。然而，两种大小显着不同的 RNA 物种（例如 Cas9 mRNA (4.5 kb) 和 sgRNA (0.1 kb)）的共传递仍然具有挑战性，尤其是在体内。在这里，我们通过使用聚乙二醇化聚合胶束 (PM) 将 RNA 浓缩在其核心来解决这个问题。单独的 PM 加载 sgRNA 在缓冲液中以最小稀释度释放 sgRNA，而单独的 PM 加载 Cas9 mRNA 即使在更高的稀释度下也是稳定的。有趣的是，将 sgRNA 与 Cas9 mRNA 共同封装在单个 PM 中可以防止稀释后 sgRNA 的释放，从而增强 sgRNA 对酶促降解的耐受性。随后，在脑实质内注射后，与共封装 RNA 一起的 PM 在小鼠大脑的实质细胞（包括神经元、星形胶质细胞和小胶质细胞）中广泛诱导基因组编辑，其效率比分别负载 Cas9 mRNA 或 sgRNA 的 PM 共同递送的效率更高。据我们所知，这是第一份证明基于 RNA 的 CRISPR/Cas9 递送在诱导脑实质细胞基因组编辑方面的效用的报告。此外，由于 PM 在脑组织中的扩散增强，使用 PM 进行基因组编辑的效率高于使用非聚乙二醇化的复合物。本文报告的结果证明了使用 PM 共封装 Cas9 mRNA 和 sgRNA 用于体内基因组编辑的潜力。
Genome editing using CRISPR/Cas9 has attracted considerable attention for the treatment of genetic disorders and viral infections. Co-delivery of Cas9 mRNA and single guide (sg)RNA is a promising strategy to efficiently edit the genome of various cell types, including non-dividing cells, with minimal safety concerns. However, codelivery of two RNA species with significantly different sizes, such as Cas9 mRNA (4.5 kb) and sgRNA (0.1 kb), is still challenging, especially in vivo. Here, we addressed this issue by using a PEGylated polyplex micelle (PM) condensing the RNA in its core. PM loading sgRNA alone released sgRNA at minimal dilution in buffer, while PM loading Cas9 mRNA alone was stable even at higher dilutions. Interestingly, co-encapsulating sgRNA with Cas9 mRNA in a single PM prevented sgRNA release upon dilution, which led to the enhanced tolerability of sgRNA against enzymatic degradation. Subsequently, PM with co-encapsulated RNA widely induced genome editing in parenchymal cells in the mouse brain, including neurons, astrocytes, and microglia, following intraparenchymal injection, at higher efficiency than that by co-delivery of PMs loaded with either Cas9 mRNA or sgRNA separately. To the best of our knowledge, this is the first report demonstrating the utility of RNA-based delivery of CRISPR/Cas9 in inducing genome editing in the brain parenchymal cells. Furthermore, the efficiency of genome editing using PMs was higher than using a non-PEGylated polyplex, due to the enhanced diffusion of PMs in the brain tissue. The results reported herein demonstrate the potential of using PMs to co-encapsulate Cas9 mRNA and sgRNA for in vivo genome editing.