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

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.