New Method Enables Efficient Full-Length Human Gene Integration in Mice

Understanding how human genes function in living systems has long been limited by differences between species. While mice share most protein-coding sequences with humans, their gene regulation often diverges, making it difficult to replicate human biology accurately. To overcome these challenges, scientists have pursued full-length gene humanization (FL-GH), where entire mouse genomic loci—including coding sequences, introns, untranslated regions, and regulatory elements—are replaced with their human equivalents. However, existing methods have struggled to insert large DNA fragments efficiently or reliably, slowing the creation of physiologically relevant humanized models.

Now, a team led by Manabu Ozawa and Jumpei Taguchi at The Institute of Medical Science, The University of Tokyo, has developed a new method called TECHNO (Two-step ES Cell-based HumaNizatiOn) to address these difficulties. Their study, published in Nature Communications, presents a two-step approach that combines CRISPR/Cas9-assisted genome editing with bacterial artificial chromosome (BAC) technology for the integration of full-length human genomic regions. “Our results demonstrate a robust and broadly applicable platform for generating FL-GH mouse models,” says Dr. Ozawa. 

TECHNO operates through two coordinated steps. First, the target mouse locus is removed using Cas9 ribonucleoproteins and replaced with short human homology arms flanking a selection cassette, creating a precise genomic landing site. In the second step, a BAC carrying the complete human gene and its regulatory components is introduced into embryonic stem cells with a universal guide RNA targeting the selection cassette. This enables homology-directed integration of genomic segments larger than 200 kilobase pairs. As the method uses standard molecular tools and accessible BAC libraries, it could apply to more than 90% of human genes.

Using this platform, the researchers humanized several loci, including c-Kit, APOBEC3, and CYBB. The c-Kit substitution reproduced human-like splicing and organ-specific expression while maintaining essential processes such as hematopoiesis and spermatogenesis. Replacing the APOBEC3 locus demonstrated large-scale integration of over 200 kbp of DNA spanning seven genes, with human-like expression patterns. A humanized CYBB allele with disease-associated mutations successfully modeled chronic granulomatous disease, producing defects in reactive oxygen species similar to those found in patients. 

As Dr. Ozawa notes, “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.”