Mouse genome rewriting and tailoring of three important disease loci [ChIP]

Genetically Engineered Mouse Models (GEMMs) aid in understanding human pathologies and developing new therapeutics, yet recapitulating human diseases authentically in mice is challenging to design and execute. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences controlling spatiotemporal gene expression patterns and splicing to human diseases. It is thus apparent that including regulatory genomic regions during the engineering of GEMMs is highly preferable for disease modeling, with the prerequisite of large-scale genome engineering ability. Existing genome engineering methods have limits on the size and efficiency of DNA delivery, hampering routine creation of highly informative GEMMs. Here, we describe mSwAP-In (mammalian Switching Antibiotic resistance markers Progressively for Integration), a method for efficient genome rewriting in mouse embryonic stem cells. We first demonstrated the use of mSwAP-In for iterative genome rewriting of up to 115 kb of the Trp53 locus, as well as for genomic humanization of up to 180 kb ACE2 locus in response to the COVID-19 pandemic. Second, we showed the hACE2 GEMM authentically recapitulated human ACE2 expression patterns and splicing, and importantly, presented milder symptoms without mortality when challenged with SARS-CoV-2 compared to the K18-ACE2 model, thus representing a more authentic model of infection. Lastly, we demonstrated serial genome writing by humanizing mouse Tmprss2 in a biallelic fashion, highlighting the versatility of mSwAP-In in mouse genome writing. Three groups (WT, 116kb-hACE2, 180kb-hACE2) of mouse testes were included in this dataset, each group has two biological replicates, being pulled down with IgG negative control, and H3K4me3 and H3K27ac antibodies.

基因工程小鼠模型（Genetically Engineered Mouse Models, GEMMs）有助于解析人类疾病病理并开发新型治疗手段，然而在小鼠体内真实重现人类疾病，在设计与实施层面均颇具挑战。基因组学领域的进展已凸显出：调控基因时空表达模式与可变剪接的非编码调控基因组序列，与人类疾病密切相关。因此，在基因工程小鼠模型构建过程中纳入调控基因组区域，显然是更优的疾病建模方案，而其前提是具备大规模基因组工程操作能力。现有基因组工程方法在DNA递送的片段长度与效率上存在局限，阻碍了高信息价值基因工程小鼠模型的常规构建。本文报道了mSwAP-In（哺乳动物整合型抗生素抗性标记逐步替换系统，mammalian Switching Antibiotic resistance markers Progressively for Integration），一种可在小鼠胚胎干细胞中实现高效基因组重写的技术方法。首先，我们验证了mSwAP-In可用于对长达115 kb的Trp53基因座进行迭代式基因组重写，同时也可针对新冠疫情需求，实现长达180 kb的ACE2基因座的基因组人源化改造。其次，我们证实，构建的hACE2基因工程小鼠模型可真实重现人类ACE2的表达模式与可变剪接形式；重要的是，与K18-ACE2模型相比，该模型在感染SARS-CoV-2后症状更轻微且无死亡案例，因此是更贴合真实感染情况的疾病模型。最后，我们通过双等位基因方式对小鼠Tmprss2基因座进行人源化改造，实现了连续多轮基因组写入，进一步凸显了mSwAP-In在小鼠基因组改造中的通用性。本数据集包含三组小鼠睾丸样本（野生型WT、116kb-hACE2、180kb-hACE2），每组设置两个生物学重复，分别采用IgG阴性对照、H3K4me3及H3K27ac抗体进行染色质免疫沉淀下拉实验。