人类散发性结直肠癌 (CRC) 是结直肠上皮中特定基因突变连续获得的多步骤途径的结果。 CRC 体内建模对于了解肿瘤微环境至关重要。为了准确地概括人类 CRC 发病机制，小鼠模型必须包括这些多步骤遗传异常。生成更接近地模拟此多步骤过程的散发 CRC 模型，并使用该模型研究新型 Let7 靶点 PLAGL2 在 CRC 发病机制中的作用。我们构建了 CRISPR/Cas9 体细胞诱变小鼠模型，该模型可诱导且多重，可同时灭活与 CRC 发病机制相关的多个基因。我们使用多西环素诱导型转录激活剂和阿霉素失活转录抑制剂来实现 Cas9 切口酶的紧密、非泄漏表达。该小鼠具有多种向导RNA的转基因表达，可诱导肠上皮中CRC、Apc、Pten、Smad4和Trp53中常见突变的四种肿瘤抑制基因的零星失活。这些小鼠与 Vil-LCL-PLAGL2 小鼠杂交，这些小鼠在肠道上皮细胞中具有 Cre 诱导的 PLAGL2 过度表达。这些小鼠在所有四种靶向肿瘤抑制基因中表现出随机体细胞突变，导致小肠和结肠中出现多发性腺瘤和腺癌。与 Vil-LCL-PLAGL2 小鼠杂交表明，肠道特异性 PLAGL2 过度表达会增加结肠肿瘤的生长。该条件模型代表了一种新的 CRISPR/Cas9 介导的结直肠癌小鼠模型。这些小鼠可用于研究新的、以前未表征的基因在 CRC 中的作用，在多个常见突变的肿瘤抑制基因的背景下，从而更接近地模拟人类 CRC 发病机制。版权所有 © 2024 作者。由爱思唯尔公司出版。保留所有权利。

Human sporadic colorectal cancer (CRC) results from a multistep pathway with sequential acquisition of specific genetic mutations in the colorectal epithelium. Modeling CRC in vivo is critical for understanding the tumor microenvironment. To accurately recapitulate human CRC pathogenesis, mouse models must include these multi-step genetic abnormalities.Generate a sporadic CRC model that more closely mimics this multi-step process and use this model to study the role of a novel Let7 target PLAGL2 in CRC pathogenesis.We generated a CRISPR/Cas9 somatic mutagenesis mouse model that is inducible and multiplexed for simultaneous inactivation of multiple genes involved in CRC pathogenesis. We used both a doxycycline-inducible transcriptional activator and a dox-inactivated transcriptional repressor to achieve tight, non-leaky expression of the Cas9 nickase. This mouse has transgenic expression of multiple guide RNAs to induce sporadic inactivation in the gut epithelium of four tumor suppressor genes commonly mutated in CRC, Apc, Pten, Smad4 and Trp53. These were crossed to Vil-LCL-PLAGL2 mice which have Cre-inducible overexpression of PLAGL2 in the gut epithelium.These mice exhibited random somatic mutations in all four targeted tumor suppressor genes, resulting in multiple adenomas and adenocarcinomas in the small bowel and colon. Crosses with Vil-LCL-PLAGL2 mice demonstrated that gut-specific PLAGL2 overexpression increased colon tumor growth.This conditional model represents a new CRISPR/Cas9-mediated mouse model of colorectal carcinogenesis. These mice can be used to investigate the role of novel, previously uncharacterized genes in CRC, in the context of multiple commonly mutated tumor suppressor genes and thus more closely mimic human CRC pathogenesis.Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.