CIRI, Centre International de Recherche en Infectiologie Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007, Lyon, France;
LBMC, Laboratoire de Biologie et Modélisation de la Cellule Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, UMR 5239, INSERM, U1210, Lyon, 69007, France.
J Vis Exp. 2021 Mar 31(169). doi: 10.3791/62245.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells. To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named "Nanoblades") were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for "transfection-free" homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.
簇状规律间隔短回文重复 (CRISPR)-Cas 系统使真核细胞的基因组编辑民主化，并导致了许多创新应用的发展。然而，将 Cas9 蛋白和单指导 RNA (sgRNA) 递送到靶细胞在技术上具有挑战性。经典的病毒载体，如来自慢病毒 (LVs) 或腺相关病毒 (AAVs) 的载体，可以有效地递送到许多原代细胞和体内编码 Cas9 蛋白及其相关 sgRNA 的转基因。然而，这些载体可能存在一些缺点，如转基因在靶细胞基因组中的整合、有限的货物容量以及 Cas9 蛋白和指导 RNA 在靶细胞中的长期表达。为了克服其中的一些问题，开发了一种基于鼠白血病病毒 (MLV) 的递送载体，用于在不携带任何编码转基因的情况下包装 Cas9 蛋白及其相关的指导 RNA。通过将 Cas9 蛋白融合到 MLV 的结构蛋白 Gag 的 C 末端，形成了装载 Cas9 蛋白和 sgRNA 的病毒样颗粒 (VLPs) (命名为"Nanoblades")。Nanoblades 可以从产生细胞的培养基中收集、纯化、定量，并用于转导靶细胞和递送活性 Cas9/sgRNA 复合物。Nanoblades 可以在广泛的原代和永生化细胞中快速瞬时递送电核糖核酸蛋白 (RNP) 货物，并且可以使用修饰的 Cas9 蛋白进行其他应用，如靶向基因的瞬时转录激活。Nanoblades 能够在注射成年小鼠的肝脏和卵母细胞中进行体内基因组编辑，以产生转基因动物。最后，它们可以与供体 DNA 复合，用于"无转染"同源定向修复。Nanoblade 的制备简单、相对成本低，并且可以在任何细胞生物学实验室中轻松进行。
