Targeted Transgenic Mice Using CRISPR /Cas9 Technology

Methods Mol Biol. 2021;2214:125-141. doi: 10.1007/978-1-0716-0958-3_9.

Abstract

CRISPR /Cas9 is a powerful technology that has transformed gene editing of mammalian genomes, being faster and more cost-effective than standard gene targeting techniques. In this chapter, we provide a step-by-step protocol to obtain Knock-Out (KO ) or Knock-In (KI ) mouse models using CRISPR /Cas9 technology. Detailed instructions for the design of single guide RNAs (sgRNA ) for KO approaches and single-strand oligonucleotide (ssODN ) matrix for generation of KI animals are included. We also describe two independent CRISPR /Cas9 delivery methods to produce gene-edited animals starting from zygote-stage embryos, based either on cytoplasmic injection or electroporation.

Keywords: CRISPR/Cas9 gene editing; Constitutive Knock-In (KI); Constitutive Knock-Out (KO); Single guide RNA (sgRNA); Single-stranded oligodeoxynucleotides (ssODN); Transgenic mouse; Zygote electroporation; Zygote microinjection.

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Electroporation / methods
  • Female
  • Gene Editing / methods
  • Gene Knock-In Techniques / methods*
  • Gene Knockout Techniques / methods*
  • Mice
  • Mice, Transgenic / genetics*
  • RNA, Guide / genetics
  • Zygote

Substances

  • RNA, Guide