A scalable two-step genome editing strategy for generating full-length gene humanized mice at diverse genomic loci

Full-length gene-humanized mice, generated by replacing mouse loci with their human counterparts including untranslated and regulatory regions, offer a powerful in vivo platform to study human gene function. However, reliable humanization of large genomic regions remains challenging due to limitations in DNA insert size, complex protocols, and reliance on specialized materials. Here, we present a streamlined approach that enables full-length gene humanization through two sequential CRISPR-assisted homologous recombination steps in embryonic stem cells. This method supports targeted knock-in of genomic fragments exceeding 200 kbp and is applicable across multiple mouse strains. Humanized alleles generated using this strategy recapitulate human-like splicing isoforms and organ-specific gene expression, and restore essential functions in hematopoiesis, spermatogenesis, and survival. Furthermore, disease-associated mutations can be introduced into humanized alleles to generate in vivo models of human genetic disorders. This versatile platform enables creation of physiologically relevant, fully gene-humanized mouse models for broad applications in biomedical research.