School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore.
NTU Institute of Health Technologies, Interdisciplinary Graduate School, Nanyang Technological University, Singapore, Singapore.
Methods Mol Biol. 2021;2211:183-191. doi: 10.1007/978-1-0716-0943-9_13.
The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are delivered in the format of plasmids, mRNA, or ribonucleoprotein (RNP) complexes. In particular, non-viral approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local delivery and provide possible synergistic effects with other factors such as topographical cues that are co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be used to validate the efficiency of gene disruption.
简单且用途广泛的 CRISPR/Cas9 系统是一种很有前途的哺乳动物细胞基因组编辑策略。通常，基因组编辑组件（即 Cas9 蛋白和单链向导 RNA(sgRNA)）以质粒、mRNA 或核糖核蛋白 (RNP) 复合物的形式递送至细胞内。特别是，非病毒方法是可取的，因为它们克服了病毒载体带来的安全问题。为了控制组织再生中的细胞命运，可以通过支架递送来控制基因组编辑组件，这将提供局部递药的途径，并与其他因素（如同时递药的拓扑线索）产生协同作用。在这一章中，我们详细介绍了一种简单的表面修饰方法，即用 CRISPR/Cas9 RNP 复合物功能化电纺纳米纤维。我们将使用贻贝类生物黏附涂层，因为它是一种将生物分子固定在表面上的简单有效的方法。纳米纤维将为接种细胞提供仿生微环境和拓扑线索。为了评估，我们将使用带有单个增强型绿色荧光蛋白 (U2OS.EGFP) 拷贝的模型细胞系来验证基因敲除的效率。
