Abstract
Various molecular biology techniques implementing genome editing have made it possible to generate mouse mutants for nearly all known genes; as a result, the International Mouse Phenotyping Consortium (IMPC) database listing the phenotypes of genetically modified mice has been established. Among mouse phenotypes, lethality is crucial to evaluate the importance of genes in mouse survival. Although many genes are reported to show “preweaning lethality, incomplete penetrance” in the IMPC database, the survival rates of homozygous knockout mice are highly variable. Here, we propose the lethal allele index (LAI), the ratio of the observed number of mice with homozygous knockout (KO) to the theoretically predicted number of homozygous KO mice, as a simple quantitative indicator of preweaning lethality. Among the mice mutants registered as incompletely lethal in IMPC, the LAI calculated from the genotypes of F1 mice tended to be lower in disease-related genes, and correlated with the frequency of loss-of-function (LOF) alleles in humans. In genome-edited mice using CRISPR/Cas9, the number of mice with homozygous frameshift alleles seemed to be associated with lethality. We edited the Ehd1 gene in cell lines as well as mice using CRISPR/Cas9, and found that the genotype distribution was significantly different. The LAI calculated from these data was similar to the value calculated from the IMPC data. These findings support the potential usefulness of the LAI as an index of preweaning lethality in genome-edited mice.
Highlights
The development of genome editing technologies has allowed for the determination of the phenotypes of genetically modified mice for all genes, and databases that accumulate mouse phenotype information, such as the International Mouse Phenotyping Consortium (IMPC, http://www.mousephenotype.org/), have been established[1]
We proposed the Lethal Allele Index (LAI) as a quantitative index for “preweaning lethality, incomplete penetrance” as the rate of the number of mice with homozygous LOF alleles to the number predicted from the genotypes of other mice
The significant difference in genotypes between Ehd[1] mutant mice and Ehd[1] mutant PC12 cell lines generated by the CRISPR/Cas[9] system suggests that the lower rate of mice homozygous for frameshift alleles is due to the lethality of homozygous LOF alleles
Summary
The development of genome editing technologies has allowed for the determination of the phenotypes of genetically modified mice for all genes, and databases that accumulate mouse phenotype information, such as the International Mouse Phenotyping Consortium (IMPC, http://www.mousephenotype.org/), have been established[1]. Compared to the CRISPR/Cas[9] system, conventional molecular biology technologies, such as gene targeting methods, require more time to produce genome-edited mice. This is due to their lower recombination efficiency, and the necessity to generate genome-edited embryonic stem (ES) cells before establishing mutant mouse lines[5,6,7]. As a proof of concept, we generated the genome-edited mice and cell lines of EH domain containing 1 (Ehd1) gene, which is classified as “preweaning lethality, incomplete penetrance”[10,11,12], using CRISPR/Cas[9], and assessed the LAI
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