Hebert Jess D, Xu Haiqing, Tang Yuning J, Ruiz Paloma A, Detrick Colin R, Wang Jing, Hughes Nicholas W, Donosa Oscar, Siah Vicky P, Andrejka Laura, Karmakar Saswati, Aboiralor Irenosen, Tang Rui, Sotillo Rocio, Sage Julien, Cong Le, Petrov Dmitri A, Winslow Monte M
Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
Department of Biology, Stanford University, Stanford, CA, USA.
Nat Biomed Eng. 2025 May 30. doi: 10.1038/s41551-025-01407-7.
Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations. However, existing models have a limited ability to create multiple targeted edits, hindering our understanding of complex genetic interactions. Here we generate transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a), which robustly generates compound genotypes, including diverse cancers driven by inactivation of trios of tumour suppressor genes or an oncogenic translocation. We integrate these modular CRISPR RNA (crRNA) arrays with clonal barcoding to quantify the size and number of tumours with each array, as well as the impact of varying the guide number and position within a four-guide array. Finally, we generate tumours with inactivation of all combinations of nine tumour suppressor genes and find that the fitness of triple-knockout genotypes is largely explainable by one- and two-gene effects. These Cas12a alleles will enable further rapid creation of disease models and high-throughput investigation of coincident genomic alterations in vivo.
小鼠模型中的体细胞基因组编辑增进了我们对基因改变体内效应的理解。然而，现有模型创建多个靶向编辑的能力有限，这阻碍了我们对复杂基因相互作用的理解。在此，我们生成了转基因小鼠，其具有经Cre调节的增强型酸氨基球菌属Cas12a（enAsCas12a）的组成型表达，该表达能强有力地产生复合基因型，包括由三组肿瘤抑制基因失活或致癌易位驱动的多种癌症。我们将这些模块化的CRISPR RNA（crRNA）阵列与克隆条形码整合，以量化每个阵列产生的肿瘤大小和数量，以及改变四向导RNA阵列内向导数量和位置的影响。最后，我们通过使九个肿瘤抑制基因的所有组合失活来生成肿瘤，并发现三敲除基因型的适应性在很大程度上可由单基因和双基因效应来解释。这些Cas12a等位基因将有助于进一步快速创建疾病模型，并在体内对同时发生的基因组改变进行高通量研究。
