Engineering Base Changes and Epitope-Tagged Alleles in Mice Using Cas9 RNA-Guided Nuclease

Abstract

Mice carrying patient-associated base changes are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence—the orthologous sequence for base changes or the N or C terminus for epitope tags—appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target sequence variation introduced by Cas9. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Single guide RNA design and synthesis

Alternate Protocol 1: Single guide RNA synthesis by primer extension and in vitro transcription

Basic Protocol 2: Design of oligonucleotide repair template

Basic Protocol 3: Preparation of RNA mixture for microinjection

Support Protocol 1: Preparation of microinjection buffer

Alternate Protocol 2: Preparation of RNP complexes for electroporation

Basic Protocol 4: Collection and preparation of mouse zygotes for microinjection or electroporation

Basic Protocol 5: Electroporation of Cas9 RNP into zygotes using cuvettes

Alternate Protocol 3: Electroporation of Cas9 RNP into zygotes using electrode slides

Basic Protocol 6: Screening and quality control of derived mice

Support Protocol 2: Deconvoluting multiple sequence chromatograms with DECODR