Mouse genome rewriting and tailoring of three important disease loci

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.

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Sample size
Sample size was not predetermined for the in vitro cell culture experiments, including synTrp53 and its subsequent genome writing, ACE2 humanization in mESCs.We reported the number of screened colonies and the number of winners for each experiment.For RT-qPCR of SARS-CoV-2 infected animals, sample size was determined by animal sample size, animal sample sizes were chosen to enable significant statistical power while minimizing unnecessary wastage.Three RT-qPCR technical replicates were performed based on the standard in the field.
For the first qualitative SARS-CoV-2 infection experiment, with the goal of identifying an optimal infection titer (10^3PFU or 10^5PFU), we minimized the animal number by using 1 male and 1 female for each genotype (2 female K18-ACE2 mice were used for the 10^3PFU group,because the male K18-ACE2 mice were wounded due to fights).For the subsequent hACE2 mouse and golden hamster infections, 4 or 5 animals were used for each group to achieve statistical significance.For the longitudinal SARS-CoV-2 infection experiment, 9 animals for each genotype were infected and 3 were sacrificed for tissue collections at each post-infection time point.For human TMPRSS2 isoform 1 and 2 detection, one animal was used for 9 tissues collection, RT-qPCR was performed with 3 technical replicates.For CUT&RUN and ATAC-seq of testicular or small intestinal cells, two animals were used as biological replicates for each genotype.When analyzing the dACE2 isoform levels in the infected and uninfected lungs, 3 uninfected mice were used as control, all the infected 116kb-hACE2 (8) were used an unpaired twotailed, Mann-Whitney t test was used.For biallelic TMPRSS2 humanization, three different founder mESC lines were used to rule out founder effect.
Data exclusions When calculating the mSwAP-In efficiency for synTrp53,two mESC clones were excluded due to multiple copies of synTrp53 payload were detected in the subsequent capture-sequencing analysis.

Replication
The mSwAP-In engineering of Trp53, ACE2 and TMPRSS2 were performed at least three times with similar success rate each time.SARS-CoV-2 infection of hACE2 mice were performed four times, each time with K18-ACE2 mice as positive infection control as well as wild-type mice as negative infection control.
Randomization Similar aged mice and hamsters were selected randomly for the infection experiments.

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April 2023

Blinding
Investigators were not blinded to infected animal groups due to data collection and analysis in the BSL3 facility.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.The C57BL/6J male mESC line (MK6) was obtained from NYU Langone Health Rodent Genetic Engineering Lab.Mouse embryonic fibroblast (MEF) cells were purchased from CellBiolabs (CBA-310), and were subsequently inactivated via mitomycin-C treatment.VeroE6 cells were purchased from ATCC (CRL-1586).

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April 2023 for the SARS-CoV-2 infection.All animals were settled for at least two days in the NYU Langone Health BSL3 facility prior to the SARS-CoV-2 infection.

Wild animals
This study did not include wild animals.

Reporting on sex
Initially we infected both male and female mice, however, we did not notice a significant difference in terms of infectibility due to sex difference.Thus, we used female mice for the following experiments.
Field-collected samples This study did not include samples collected from the field.

Ethics oversight
All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at NYU Langone Health.
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