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 for 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. However, the editing conditions and protocols to produce knockout lines have been optimised for a limited number of strains or stocks. Here, we demonstrate a novel and universal approach for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in 8 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 novel approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research. Summary statement Universal method for zygote genome editing in multiple inbred mouse strains allows for generation of novel mutant mice for understanding mammalian gene function and human disease pathogenesis.