Mouse genome rewriting and tailoring of three important disease loci

Abstract

Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe ‘mammalian switching antibiotic resistance markers progressively for integration’ (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.

Writing the dark matter of the human genome into mice to better replicate human disease

“…Mice are naturally resistant to SARS-CoV-2; whereas a previously made engineered human ACE2 mouse model expressing too much ACE2 protein on the cell surface resulted in the mice dying too fast from COVID ( 2 ). In contrast, the newly engineered mouse model had mild infection symptoms more consistent with those observed in humans ( 1 ). Moreover, the cell and tissue expression patterns more accurately reflected those found in humans, with ACE2 now found in the mouse testis and expressed at lower levels in the lung.…”

“…Now, Zhang et al. ( 1 ), working in Jef Boeke’s group at New York University Grossman School of Medicine in the USA, developed a way to study these human regulatory codes by genetically ‘writing’ as much as 180 kilobases of human DNA in place of the corresponding mouse regions in mouse embryonic stem (ES) cells.…”

Application and Technical Challenges in Design, Cloning, and Transfer of Large DNA

“…For example, during the COVID-19 pandemic, the generation of the SARS-CoV-2 genome has the potential to promote the unraveling of disease mechanisms and the development of vaccines [88]. Zhang et al [31] constructed genetically engineered mouse models by introducing 116 kb and 180 kb humanized ACE2 loci in S. cerevisiae, which, when compared to the existing K18-hACE2 models, presented milder symptoms upon exposure to SARS-CoV-2 and more closely resembled human infection models.…”

Pathogenesis and virulence of coronavirus disease: Comparative pathology of animal models for COVID-19

“…To better replicate human ACE2 expression dynamics in mice, Zhang et al [ 129 ] recently reported a novel genome writing method to generate humanized ACE2 mice. These mice express the regulatory components and splice isoforms of human ACE2 at levels that mimic expression in people, so are potentially a more physiologically relevant mouse model of COVID-19.…”