Sandoval Ivette M, Collier Timothy J, Manfredsson Fredric P
Department of Translational Science & Molecular Medicine, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA.
Mercy Health Saint Mary's, Grand Rapids, MI, USA.
Methods Mol Biol. 2019;1937:29-45. doi: 10.1007/978-1-4939-9065-8_2.
Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) system has emerged as an extremely useful tool for biological research and as a potential technology for gene therapy approaches. CRISPR/Cas mediated genome editing can be used to easily and efficiently modify endogenous genes in a large variety of cells and organisms. Furthermore, a modified version of the Cas9 nuclease has been developed that can be used for regulation of endogenous gene expression and labeling of genomic loci, among other applications. This chapter provides an introduction to the basis of the technology and a detail protocol for the most classic application: gene inactivation by CRISPR/Cas9 nuclease system from Streptococcus pyogenes. This workflow can be easily adapted for other CRISPR systems and applications.
成簇规律间隔短回文重复序列（CRISPR/Cas）系统已成为生物学研究中极为有用的工具，也是基因治疗方法的一项潜在技术。CRISPR/Cas介导的基因组编辑可用于轻松、高效地修饰多种细胞和生物体中的内源基因。此外，已经开发出一种经过修饰的Cas9核酸酶版本，可用于内源基因表达的调控以及基因组位点的标记等其他应用。本章介绍了该技术的基础，并详细说明了最经典应用的方案：利用化脓性链球菌的CRISPR/Cas9核酸酶系统进行基因失活。该工作流程可轻松适用于其他CRISPR系统和应用。
