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. Overall design: Refer to individual Series

通过将小鼠基因组位点替换为包含非翻译区与调控区域的人类同源序列所构建的全长基因人源化小鼠，为研究人类基因功能提供了强有力的体内研究平台。然而，由于DNA插入片段尺寸限制、实验流程复杂以及依赖特殊实验材料，对大片段基因组区域进行可靠的人源化改造仍存在诸多挑战。本研究报道了一种简化的实验策略，可通过胚胎干细胞中两步连续的CRISPR（成簇规律间隔短回文重复序列）辅助同源重组步骤实现全长基因的人源化改造。该方法可实现200千碱基对以上基因组片段的靶向敲入，且适用于多种小鼠品系。采用该策略构建的人源化等位基因可重现类人类的剪接异构体与器官特异性基因表达模式，并能恢复造血、精子发生及生存所需的核心生物学功能。此外，可将疾病相关突变引入人源化等位基因中，从而构建人类遗传性疾病的体内模型。这一多用途平台可构建具有生理相关性的完全基因人源化小鼠模型，适用于生物医学研究的广泛应用场景。整体实验设计：详见各独立数据集系列。