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. 2020 Mar;10(1):e69.
doi: 10.1002/cpmo.69.

Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents

Almudena Fernández  1   2 , Matías Morín  2   3 , Diego Muñoz-Santos  1   2 , Santiago Josa  1   2 , Andrea Montero  1   2 , Marcos Rubio-Fernández  1   2 , Marta Cantero  1   2 , Julia Fernández  1   2 , María Jesús Del Hierro  1 , Marta Castrillo  1 , Miguel Ángel Moreno-Pelayo  2   3 , Lluís Montoliu  1   2
Affiliations

Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents

Almudena Fernández et al. Curr Protoc Mouse Biol. 2020 Mar.

Abstract

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.

Keywords: CRISPR; animal model; cytoplasmic injection and electroporation; genome editing; in vivo gene editing; mouse model; next-generation sequencing.

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References

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