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.

Keywords: Cas9; electroporation; epitope tags; genome editing; microinjection; mouse; single nucleotide variant.

Figure 1

Schematic workflow for generating base‐change or epitope‐tagged alleles in mice using Cas9 endonuclease and single‐strand DNA oligonucleotide repair templates. Step 1. Allele design. Design the desired allele, select the sgRNA protospacer and design the repair template and genotyping PCR assays. Step 2. Acquire reagents. Order the required primers and repair template and synthesize sgRNA. Timing for this step depends on the lead time for ordering reagents. Step 3. Prepare embryo donors and recipients. After reagents are ready, embryo donors are superovulated (start 3 days before embryo treatment) and pseudopregnant recipients are prepared (start 1 day before embryo treatment). Superovulated embryo donors are mated to fertile stud males the day before the experiment. The number of embryos donors required is strain dependent; we typically use 10 embryo donors from C57BL/6J or C57BL6/N mice to obtain 80‐120 zygotes for endonuclease treatment and prepare 6 pseudopregnant recipients. The timeline does not account for lead time to acquire embryo donors and prepare stud males. Step 4. Treat and transfer embryos. Prepare the endonuclease mix and keep on ice until embryo treatment. Collect embryos. Electroporate or microinject zygotes according to the appropriate protocol. Transfer ∼20‐25 viable embryos into each pseudopregnant recipient. Step 5. Screen founders. Isolate DNA from pup tissue biopsies, perform PCR, and sequence amplicons to identify founders with the desired allele. DECODR analysis can assist in identifying founders. Step 6. Germline transmission test breeding. At breeding age, mate each founder to a wild‐type mouse from the desired strain background. We typically test cross up to 3 founders and hold additional founders as “backups” until allele germline transmission is confirmed. The timeline in this figure assumes germline transmission in the first litter. We have often obtained germline transmission when only one or two founders were obtained and available for breeding. Step 7. Genotype N1 mice. Genotype mice born from test crosses using the same assays used to identify founders. All N1 mice that carry the allele should be heterozygous (hemizygous in the case of males born from X‐linked gene editing experiments). If germline transmission does not occur in the first litter(s), we screen at least 28 pups from each test cross before setting up additional test crosses or repeating the experiment. Step 8. Quality control N1 mice. Use at minimum copy number assessment of the template to QC N1 mice. Subcloning the PCR amplicons from genotyping provides confirmation of the allele sequence. We recommend designing one‐step real‐time allelic discrimination assays (e.g., Taqman or equivalent assays) or tag‐anchored PCR assays for routine genotyping rather than PCR and sequencing. Created using BioRender.

Figure 2

DECODR analysis can assist with identifying correctly tagged alleles in mosaic founders. (A) DECODR output for four founders’ ab1 sequence files. Boxed bases indicate nucleotides different from the wild‐type sequence. In this example we expected a 48‐bp insertion. DECODR identified a 48‐bp insertion in founder 1 and only partial insertions in the other three founders. (B) More detailed DECODR output (obtained by clicking on the file name shown in (A)) indicates that in founder 1, 45.7% of the chromatograms match the HDR (template) sequence and 54.3% of the chromatograms have a single base insertion at the endonuclease cut site. (C) The sequence alignment (SnapGene software) of founder 1's ab1 file to the desired tagged allele sequence. In the DECODR output, the green bar indicates the gRNA spacer sequence; the red bar indicates the PAM; and the pipe (vertical line) in the sequence indicates the predicted endonuclease cut site. Created in BioRender.