Two-Step CRISPR Enables Full-Length Mouse Gene Humanisation

“This study introduces a streamlined approach that enables full-length gene humanisation through two sequential CRISPR-assisted homologous recombination steps in embryonic stem cells”Jumpei Taguchi et al.

The TECHNO method addresses these limitations through two sequential CRISPR-Cas9-assisted homologous recombination steps in mouse embryonic stem cells (see Figure 1). In the first step, researchers remove the target mouse locus using CRISPR-Cas9 ribonucleoproteins and simultaneously integrate homology arms flanking the corresponding human gene sequence, along with a neomycin resistance cassette for selection.

»This method supports targeted knock-in of genomic fragments (>200 kbp) and is applicable across multiple mouse strains,« the authors note in their Nature Communications paper.

“H3K9me3, which is deposited shortly after fertilization, is required for the subsequent de novo DNA methylation at the H19-DMR”Takuro Horii et al.

The platform's utility for disease modelling was demonstrated by humanising the X-linked Cybb gene (approximately 55 kilobases), which encodes the NADPH oxidase component responsible for reactive oxygen species production in phagocytes. Following successful humanisation of the Cybb locus, the researchers introduced two chronic granulomatous disease-associated mutations (T458G and A461Δ) into the human CYBB allele via additional genome editing.

Upon phorbol myristate acetate stimulation, granulocytes from wild-type and humanised mice carrying the normal human allele generated comparable reactive oxygen species levels, whereas cells from mice carrying the mutant allele failed to increase reactive oxygen species production – precisely recapitulating the biochemical defect underlying chronic granulomatous disease in human patients.

»Overall, these results demonstrate that our method enables not only FL-GH of individual loci but also precise modelling of human genetic diseases in vivo by introducing disease-associated mutations into humanised alleles,« the authors conclude.

The authors acknowledge that whilst their approach captures complete gene structures including proximal regulatory regions, it may not encompass all distal regulatory elements, such as distant enhancers. They note that some aspects of gene expression differed between humanised mice and humans, exemplified by reduced APOBEC3A expression and the absence of detectable APOBEC3A protein in humanised mouse leukocytes. These discrepancies likely reflect fundamental interspecies differences in transcriptional regulation that cannot be overcome through simple locus replacement. Additionally, large-scale genomic humanisation can potentially influence neighbouring gene expression, as evidenced by reduced Cbx6 mRNA levels in lungs of APOBEC3-humanised mice.

The study was conducted by Jumpei Taguchi and Manabu Ozawa at the University of Tokyo. It was published in Nature Communications on 14 January 2026.