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.