Abstract—CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.

摘要： CRISPR/Cas系统是对遗传物质进行靶向操作的前景分子工具，如基因编辑、基因转录调控、表观基因组修饰等。而CRISPR/Cas系统被证明对于纠正遗传疾病和治疗传染病非常有效。和癌症的实验环境中，由于缺乏有效的 CRISPR/Cas 递送载体，这些结果的临床转化受到阻碍。基于有机和无机聚合物的现代合成纳米载体有许多缺点，包括毒性问题、缺乏靶向递送以及复杂且昂贵的生产管道。反过来，外泌体是分泌的生物纳米颗粒，具有高生物相容性、物理化学稳定性和跨越生物屏障的能力。早期临床试验未发现与外泌体注射相关的毒性。近年来，外泌体被认为是体内 CRISPR/Cas 系统的前景递送载体。本研究的目的是分析 CRISPR/Cas 随机包装到外泌体中对几种人类细胞系的功效。在这里，我们表明 Cas9 蛋白有效地定位到细胞内外泌体生物发生的区室中，但 Cas9 随机包装到外泌体中的概率变得非常低 (~1%)。因此，Cas9 蛋白的随机包装非常无效，不能用于基因编辑目的。需要开发新的工具和技术将 CRISPR/Cas 系统加载到外泌体中。