Fernández Almudena, Morín Matías, Muñoz-Santos Diego, Josa Santiago, Montero Andrea, Rubio-Fernández Marcos, Cantero Marta, Fernández Julia, Del Hierro María Jesús, Castrillo Marta, Moreno-Pelayo Miguel Ángel, Montoliu Lluís
National Centre for Biotechnology (CNB), CSIC, Madrid, Spain.
CIBER of Rare Diseases (CIBERER), ISCIII, Madrid, Spain.
Curr Protoc Mouse Biol. 2020 Mar;10(1):e69. doi: 10.1002/cpmo.69.
The simple protocol described in this article aims to provide all required information, as a comprehensive, easy-to-follow step-by-step method, to ensure the generation of the expected genome-edited mice. Here, we provide protocols for the preparation of CRISPR-Cas9 reagents for microinjection and electroporation into one-cell mouse embryos to create knockout or knock-in mouse models, and for genotyping the resulting offspring with the latest innovative next-generation sequencing methods. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategy Basic Protocol 2: Preparing and validating CRISPR-Cas9 reagents Basic Protocol 3: Preparing and injecting CRISPR-Cas9 compounds into fertilized mouse oocytes Basic Protocol 4: Genotyping genome-edited mice Support Protocol: Genotyping for CRISPR-generated "indel" mutations.
本文中描述的简单方案旨在提供所有所需信息，作为一种全面、易于遵循的分步方法，以确保生成预期的基因编辑小鼠。在此，我们提供了用于制备CRISPR-Cas9试剂的方案，以便将其显微注射和电穿孔到单细胞小鼠胚胎中，从而创建敲除或敲入小鼠模型，并使用最新的创新型下一代测序方法对所得后代进行基因分型。© 2020约翰威立国际出版公司。基本方案1：为您的基因破坏/编辑策略设计最佳RNA向导 基本方案2：制备和验证CRISPR-Cas9试剂 基本方案3：将CRISPR-Cas9化合物制备并注射到受精小鼠卵母细胞中 基本方案4：对基因编辑小鼠进行基因分型 支持方案：对CRISPR产生的“插入缺失”突变进行基因分型。
