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

Curr Protoc Mouse Biol. 2020 Mar;10(1):e69. doi: 10.1002/cpmo.69.

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.

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Gene Editing / methods*
  • Genotyping Techniques / instrumentation
  • Genotyping Techniques / methods*
  • Indicators and Reagents
  • Mice
  • Mice, Transgenic
  • Models, Animal*

Substances

  • Indicators and Reagents