Moravec Cara E, Pelegri Francisco J
Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, USA.
Methods Mol Biol. 2019;1920:377-392. doi: 10.1007/978-1-4939-9009-2_23.
The ability to create targeted mutations in specific genes, and therefore a loss-of-function condition, provides essential information about their endogenous functions during development and homeostasis. The discovery that CRISPR-Cas9 can target specific sequences according to base-pair complementarity and readily create knockouts in a desired gene has elevated the implementation of genetic analysis in numerous organisms. As CRISPR-Cas9 has become a powerful tool in a number of species, multiple methods for designing, creating, and screening editing efficiencies have been published, each of which has unique benefits. This chapter presents a cost-efficient, accessible protocol for creating knockout mutants in zebrafish using insertions/deletions (INDELS), from target site selection to mutant propagation, using basic laboratory supplies. The presented approach can be adapted to other systems, including any vertebrate species.
在特定基因中产生靶向突变并由此导致功能丧失状态的能力，为研究这些基因在发育和体内平衡过程中的内源性功能提供了重要信息。CRISPR-Cas9能够根据碱基对互补性靶向特定序列并轻易地在目标基因中产生基因敲除，这一发现提升了众多生物体中遗传分析的应用。随着CRISPR-Cas9在许多物种中成为一种强大的工具，已发表了多种设计、创建和筛选编辑效率的方法，每种方法都有其独特的优势。本章介绍了一种经济高效、易于操作的方案，该方案利用插入/缺失（INDELS）在斑马鱼中创建基因敲除突变体，从靶点选择到突变体繁殖，仅使用基本的实验室用品。所介绍的方法可适用于其他系统，包括任何脊椎动物物种。
