A universal method for generating knockout mice in multiple genetic backgrounds using zygote electroporation

Abstract

Genetically engineered mouse models are essential tools for understanding mammalian gene functions and disease pathogenesis. Genome editing allows the generation of these models in multiple inbred strains of mice without backcrossing. Zygote electroporation dramatically removed the barrier for introducing the CRISPR-Cas9 complex in terms of cost and labour. Here, we demonstrate that the generalised zygote electroporation method is also effective for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in eight common inbred strains. Long-read sequencing analysis detected not only intended mutant alleles but also differences in read frequency of intended and unintended alleles among strains. Successful germline transmission of knockout alleles demonstrated that our approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research.

Keywords: In vitro fertilisation; Electroporation; Genome editing; Knockout; Long-read sequencing; Mouse.

Fig. 1.

Genome editing and genotyping design on Hr gene. Orange arrows (gRNA-1 and gRNA-2) represent the gRNA target sites flanking exon 3. Blue arrows represent PCR primers, including the size of the PCR amplicon for G0 genotyping. Allele A represents the wild-type allele; allele B represents the exon deletion allele. gRNA, guide RNA.

Fig. 2.

Phenotype of G0 mice in various inbred strains. Mice with induced mutations in the Hr gene begin to lose hair at ∼3 weeks of age, when the second hair cycle begins. Complete loss of function of the Hr gene results in mice with complete hair loss.

Fig. 3.

DAJIN report on the allele percentage. (A) DAJIN report on the allele percentage in the eight strains. Mouse strains are indicated on the x-axis. The percentages of reads with DAJIN-predicted allele types are indicated on the y-axis. The bar colour indicates each of DAJIN predicted allele types, including intended deletion, potential wild-type, inversion and large rearrangement. The horizontal dotted line represents 10% allele percentage. Asterisks denote G0 mice used in the creation of G1 mice. (B) Scatter plots of the percentage of reads on the intended deletion and large rearrangement alleles. Dots represent each mouse sample. Mouse strains are indicated by the different colours. The percentages of reads with DAJIN-predicted allele types are indicated on the y-axis.

Fig. 4.

Heritability of genome editing mutations to progenies. (A) The number, genotype and sex of G1 mice of eight inbred strains are shown. The outer layer represents sex (blue for male and red for female), the middle layer represents the allele carried by each G1 mouse, and the inner layer shows G0 male mice used for each strain. Unread* alleles might include potential wild-type, inversion or large rearrangement alleles (not confirmed). The presence of these alleles was confirmed by electrophoresis of genomic PCR products. Note that all biological mothers of G1 mice were wild type. (B) Read percentages of mutant alleles harboured by each father of G1 mice based on DAJIN analysis. The bar colour links to the colour of each allele in G1 mice in A. Dark grey indicates unread* alleles that were not selected for detailed analysis in the genotyping of G1 mice.