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使用树枝状聚合物纳米颗粒实现CRISPR-Cas9核糖核蛋白的高效细胞内递送以进行强大的基因组编辑

Efficient Intracellular Delivery of CRISPR-Cas9 Ribonucleoproteins Using Dendrimer Nanoparticles for Robust Genomic Editing.

作者信息

Liyanage Wathsala, Kannan Gokul, Kannan Sujatha, Kannan Rangaramanujam M

机构信息

Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.

Department of Bioengineering, Stanford University School of Medicine, Stanford, CA 94305, USA.

出版信息

Nano Today. 2025 Apr;61. doi: 10.1016/j.nantod.2025.102654. Epub 2025 Jan 31.

Abstract

CRISPR-Cas9, a flexible and efficient genome editing technology, is currently limited by the challenge of delivering the large ribonucleoprotein complex intracellularly and into the nucleus. Existing delivery techniques/vectors are limited by their toxicity, immunogenicity, scalability, and lack of specific cell-targeting ability. This study presents a neutral, non-toxic dendrimer conjugate construct that shows promise in overcoming these limitations. We covalently-conjugated S. pyogenes Cas9-2NLS (Cas9-nuclear localization sequence) endonuclease to a hydroxyl PAMAM dendrimer through a glutathione-sensitive disulfide linker highly specific inverse Diels-alder click reaction (IEDDA), and a single guide RNA (sgRNA) was complexed to the Cas9-dendrimer conjugate nano-construct (D-Cas9). D-Cas9- RNP produces robust genomic deletion of GFP in HEK293 cells (~100%) and VEGF in a human pigmental epithelium cell line (ARPE-19) (20%). The uptake of the D-Cas9-RNP constructs on similar timescales as small molecules highlights the robustness of the biophysical mechanisms enabling the dendrimer to deliver payloads as large as Cas9, while retaining payload functionality. This promising conjugation approach enabled better stability to the neutral construct. Combined with recent advances in hydroxyl dendrimer delivery technologies in the clinic, this approach may lead to advances in 'neutral' dendrimer-enabled non-toxic, cell-specific, highly efficient and genome editing.

摘要

CRISPR-Cas9是一种灵活高效的基因组编辑技术,目前受到将大型核糖核蛋白复合物导入细胞内和细胞核这一挑战的限制。现有的递送技术/载体存在毒性、免疫原性、可扩展性方面的局限,且缺乏特异性细胞靶向能力。本研究提出了一种中性、无毒的树枝状聚合物共轭构建体,该构建体在克服这些限制方面显示出前景。我们通过谷胱甘肽敏感的二硫键连接子、高度特异性的逆狄尔斯-阿尔德点击反应(IEDDA),将化脓性链球菌Cas9-2NLS(Cas9-核定位序列)核酸内切酶共价连接到羟基化聚酰胺-胺树枝状聚合物上,并将单导向RNA(sgRNA)与Cas9-树枝状聚合物共轭纳米构建体(D-Cas9)复合。D-Cas9核糖核蛋白在HEK293细胞中对绿色荧光蛋白产生强大的基因组缺失(约100%),在人色素上皮细胞系(ARPE-19)中对血管内皮生长因子产生20%的基因组缺失。D-Cas9核糖核蛋白构建体在与小分子相似的时间尺度上的摄取,突出了生物物理机制的稳健性,使树枝状聚合物能够递送如Cas9这般大的有效载荷,同时保留有效载荷的功能。这种有前景的共轭方法使中性构建体具有更好的稳定性。结合临床上羟基树枝状聚合物递送技术的最新进展,这种方法可能会推动“中性”树枝状聚合物实现无毒、细胞特异性、高效的基因组编辑取得进展。

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