Department of Chemistry, Technical University of Denmark, 2800 Lyngby, Denmark.
School of Medicine, Stanford University, Stanford, CA 94350, USA.
Mol Ther. 2022 Jan 5;30(1):32-46. doi: 10.1016/j.ymthe.2021.06.003. Epub 2021 Jun 4.
CRISPR-Cas9 is rapidly entering molecular biology and biomedicine as a promising gene-editing tool. A unique feature of CRISPR-Cas9 is a single-guide RNA directing a Cas9 nuclease toward its genomic target. Herein, we highlight new approaches for improving cellular uptake and endosomal escape of CRISPR-Cas9. As opposed to other recently published works, this review is focused on non-viral carriers as a means to facilitate the cellular uptake of CRISPR-Cas9 through endocytosis. The majority of non-viral carriers, such as gold nanoparticles, polymer nanoparticles, lipid nanoparticles, and nanoscale zeolitic imidazole frameworks, is developed with a focus toward optimizing the endosomal escape of CRISPR-Cas9 by taking advantage of the acidic environment in the late endosomes. Among the most broadly used methods for in vitro and ex vivo ribonucleotide protein transfection are electroporation and microinjection. Thus, other delivery formats are warranted for in vivo delivery of CRISPR-Cas9. Herein, we specifically revise the use of peptide and nanoparticle-based systems as platforms for CRISPR-Cas9 delivery in vivo. Finally, we highlight future perspectives of the CRISPR-Cas9 gene-editing tool and the prospects of using non-viral vectors to improve its bioavailability and therapeutic potential.
CRISPR-Cas9 作为一种有前途的基因编辑工具，正在迅速进入分子生物学和生物医学领域。CRISPR-Cas9 的一个独特特点是单指导 RNA 引导 Cas9 核酸酶靶向其基因组目标。本文重点介绍了提高 CRISPR-Cas9 细胞摄取和内涵体逃逸的新方法。与其他最近发表的作品不同，本综述侧重于非病毒载体作为通过内吞作用促进 CRISPR-Cas9 细胞摄取的手段。大多数非病毒载体，如金纳米粒子、聚合物纳米粒子、脂质纳米粒子和纳米沸石咪唑酯骨架，都是为了优化 CRISPR-Cas9 的内涵体逃逸而开发的，利用晚期内涵体中的酸性环境。在体外和体内核糖核苷酸蛋白转染中最广泛使用的方法是电穿孔和显微注射。因此，需要其他输送形式来进行 CRISPR-Cas9 的体内输送。在这里，我们特别修订了基于肽和纳米粒子的系统作为 CRISPR-Cas9 体内递送平台的使用。最后，我们强调了 CRISPR-Cas9 基因编辑工具的未来展望以及利用非病毒载体提高其生物利用度和治疗潜力的前景。
