Supramolecular Nanosubstrate-Mediated Delivery for CRISPR/Cas9 Gene Disruption and Deletion.
Supramolecular Nanosubstrate-Mediated Delivery for CRISPR/Cas9 Gene Disruption and Deletion.
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DOI：
10.1002/smll.202100546
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发表时间：
2021-07
期刊：
影响因子：
--
通讯作者：
Tseng HR
中科院分区：
文献类型：
--
作者：
Ban Q;Yang P;Chou SJ;Qiao L;Xia H;Xue J;Wang F;Xu X;Sun N;Zhang RY;Zhang C;Lee A;Liu W;Lin TY;Ko YL;Antovski P;Zhang X;Chiou SH;Lee CF;Hui W;Liu D;Jonas SJ;Weiss PS;Tseng HR
CRISPR/Cas9是一种高效精确的基因编辑技术，为建立针对遗传疾病的治疗方法提供了多功能解决方案。目前CRISPR/Cas9向细胞中的递送主要依赖于病毒载体，其在包装能力和安全性方面受到限制。为了解决这些问题，我们报道了一种非病毒递送，其中Cas9·sgRNA核糖核蛋白（RNP）可以被封装到超分子纳米颗粒（SMNP）载体中以形成RNP包裹的SMNP，然后可以通过超分子纳米底物介导的递送（SNMD）策略将其递送到靶向细胞中。利用U87胶质母细胞瘤细胞系作为模型系统，我们检查了各种参数的细胞摄取的RNP负载的纳米粒子。我们进一步研究了在表达绿色荧光蛋白（GFP）的U87细胞系（GFP-U87）中剂量依赖性和时间依赖性CRISPR/Cas9介导的基因破坏。最后，我们证明了这种优化的SMNP制剂在共递送Cas9蛋白和靶向肌营养不良蛋白基因的外显子45-55（708 kb）缺失的两种sgRNA中的效用。该区域的突变导致杜氏肌营养不良症（DMD），一种严重的遗传性肌肉萎缩症。我们观察到这些基因缺失货物在人心肌细胞系（AC 16）、诱导多能干细胞（iPSC）和间充质干细胞（MSC）中的有效递送。开发了超分子纳米底物介导的递送（SNMD）策略以提高Cas9核糖核蛋白（RNP）向靶细胞中的递送效率。该策略通过分子识别将超分子纳米颗粒（SMNP）从周围介质局部富集到纳米线上，从而实现高效的CRISPR/Cas9基因破坏和缺失。该平台为DMD等遗传性疾病提供了通用的临床治疗解决方案。
CRISPR/Cas9 is an efficient and precise gene editing technology that offers a versatile solution for establishing treatments directed at genetic diseases. Current CRISPR/Cas9 delivery into cells relies primarily on viral vectors, which suffer from limitations in packaging capacity and safety concerns. To address these issues, we report a nonviral delivery where Cas9•sgRNA ribonucleoprotein (RNP) can be encapsulated into supramolecular nanoparticle (SMNP) vectors to form RNP⊂SMNPs, which can then be delivered into targeted cells via a supramolecular nanosubstrate-mediated delivery (SNMD) strategy. Utilizing the U87 glioblastoma cell line as a model system, we examine a variety of parameters for cellular-uptake of the RNP-laden nanoparticles. We further examine dose-dependent and time-dependent CRISPR/Cas9-mediated gene disruption in a green fluorescent protein (GFP)-expressing U87 cell line (GFP-U87). Finally, we demonstrate the utility of this optimized SMNP formulation in co-delivering Cas9 protein and two sgRNAs that target deletion of exons 45-55 (708 kb) of the dystrophin gene. Mutations in this region lead to Duchenne muscular dystrophy (DMD), a severe genetic muscle wasting disease. We observe efficient delivery of these gene deletion cargoes in a human cardiomyocyte cell line (AC16), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs). A supramolecular nanosubstrate-mediated delivery (SNMD) strategy is developed to improve the delivery efficiency of Cas9 ribonucleoprotein (RNP) into target cells. This strategy leverages local enrichment of supramolecular nanoparticles (SMNPs) from the surrounding medium onto nanowires via molecular recognition, enabling high-efficient CRISPR/Cas9 gene disruption and deletion. This platform offers a general clinical therapeutic solution for genetic diseases, such as DMD.
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