Two-step genome editing enables the creation of full-length humanized mouse models

Understanding human gene function in living organisms has long been hampered by fundamental differences between species. Although mice share most protein-coding genes with humans, their regulatory landscapes often diverge, limiting how accurately mouse models can mimic human biology. One promising solution is full-length gene humanization (FL-GH), in which entire mouse loci—including coding sequences, introns, untranslated regions, and regulatory elements—are replaced with their human counterparts. Yet existing technologies have struggled to insert very large genomic fragments efficiently or reliably, slowing efforts to develop physiologically relevant humanized models.

To address these long-standing challenges, a research team led by Associate Professor Manabu Ozawa and Associate Professor Jumpei Taguchi from The Institute of Medical Science, The University of Tokyo, Japan, has developed a streamlined two-step strategy for FL-GH. Their study, published in Nature Communications on January 14, 2026, introduces TECHNO (Two-step ES Cell-based HumaNizatiOn), a method that integrates CRISPR/Cas9-assisted genome editing with bacterial artificial chromosome (BAC)-based delivery of large human genomic regions. This framework offers a practical and scalable solution for replacing entire mouse loci with their human counterparts. “Our results demonstrate a robust and broadly applicable platform for generating FL-GH mouse models,” says Dr. Ozawa.

The TECHNO workflow unfolds in two coordinated steps. First, the target mouse locus is excised using Cas9 ribonucleoproteins and replaced with short human homology arms surrounding a selection cassette, creating a precise genomic landing site. In the second step, a BAC carrying the full-length human gene and its regulatory elements is introduced into embryonic stem cells alongside a universal gRNA targeting the selection cassette, enabling homology-directed integration of genomic fragments exceeding 200 kbp. Because the method relies on standard molecular reagents and widely available BAC libraries, it is theoretically applicable to more than 90% of human genes.

Using this platform, the team successfully humanized several loci, including c-Kit, APOBEC3, and CYBB. Humanization of c-Kit reproduced human-like alternative splicing and organ-specific expression while supporting essential biological functions such as hematopoiesis and spermatogenesis. Replacement of the APOBEC3 locus demonstrated the scalability of the approach, integrating over 200 kbp of human DNA spanning seven genes and generating expression patterns that mirrored those observed in humans. The researchers also established a humanized CYBB allele and introduced disease-associated mutations to model chronic granulomatous disease. The resulting mice displayed impaired reactive oxygen species production, faithfully recapitulating the molecular phenotype found in patients.

In the near term, TECHNO is expected to accelerate the development of precise, human-relevant animal models for evaluating therapeutic targets, validating disease-associated variants, and identifying ineffective drug candidates earlier in research pipelines. Over the longer term, scalable FL-GH may reshape biomedical research by enabling models that more faithfully mimic human gene regulation and disease mechanisms. These advances also set the stage for integrating humanized models into AI-driven comparative genomics, large-scale humanized allele panels, and systems biology frameworks. As Dr. Ozawa states, “Overall, these results demonstrate that our method enables not only FL-GH of individual loci but also precise modeling of human genetic diseases in vivo by introducing disease-associated mutations into humanized alleles.”

By enabling stable, high-efficiency integration of genomic fragments exceeding 200 kbp while preserving complex regulatory behavior in vivo, the TECHNO platform represents a major advance toward next-generation humanized mouse models. Its versatility, robustness, and reliance on standard laboratory tools position it as a foundational technology for advancing functional genomics, disease modeling, and translational medicine.

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Reference
Authors: Jumpei Taguchi¹, Mio Kikuchi¹, Hyojung Jeon², Ryo Shimizu³, Hideto Mori⁴, Masahito Ikawa^5,6, Yasuhiro Yamada⁷, Kei Sato⁸, Terumasa Ikeda³, Satoshi Yamazaki^1,2, and Manabu Ozawa^1,6
DOI: 10.1038/s41467-025-67900-4
Affiliations: ¹Core Laboratory for Developing Advanced Animal Models, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, The University of Tokyo, Japan
²Division of Cell Regulation, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, The University of Tokyo, Japan
³Division of Molecular Virology and Genetics, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Japan
⁴Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), The University of Osaka, Japan
⁵Department of Experimental Genome Research, Research Institute for Microbial Diseases, The University of Osaka, Japan
⁶Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, The University of Tokyo, Japan
⁷Department of Molecular Pathology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Japan
⁸Division of Systems Virology, Department of Microbiology and Immunology, Institute of Medical Science, The University of Tokyo, Japan

About The Institute of Medical Science, The University of Tokyo
The Institute of Medical Science, The University of Tokyo (IMSUT), established in 1892 as the Institute of Infectious Diseases and renamed IMSUT in 1967, is a leading research institution with a rich history spanning over 130 years. It focuses on exploring biological phenomena and disease principles to develop innovative strategies for disease prevention and treatment. IMSUT fosters a collaborative, interdisciplinary research environment and is known for its work in genomic medicine, regenerative medicine, and advanced medical approaches like gene therapy and AI in healthcare. It operates core research departments and numerous specialized centers, including the Human Genome Center and the Advanced Clinical Research Center, and is recognized as Japan’s only International Joint Usage/Research Center in life sciences.

About Associate Professor Manabu Ozawa from The Institute of Medical Science, The University of Tokyo
Associate Professor Manabu Ozawa is a developmental and reproductive biologist at The Institute of Medical Science, The University of Tokyo. His research focuses on germ-cell development, spermatogenesis, and genome engineering using embryonic stem cells and genetically modified mouse models. He specializes in large-scale genomic manipulation, including full-length gene humanization, to investigate how human genes function in vivo. Through combining advanced CRISPR technologies with ES-cell-based approaches, his work aims to create physiologically relevant models for studying human genetic disorders, developmental mechanisms, and reproductive biology. Dr. Ozawa’s contributions support translational research by enabling precise functional analysis of human genes within living organisms.

Funding information
This study was supported by T. Ando in Pathology Core laboratory, The Institute of Medical Science (IMSUT), The University of Tokyo, JSPS KAKENHI (23K27084), JSPS KAKENHI (25K18393), 2024 Inamori Research Grants, and JSPS KAKENHI (21H05033).