成年干细胞衍生器官样体中通过碱基编辑器多重复合生成肿瘤模型的一步法。
One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids.
发表日期：2023 Aug 17
作者：
Maarten H Geurts, Shashank Gandhi, Matteo G Boretto, Ninouk Akkerman, Lucca L M Derks, Gijs van Son, Martina Celotti, Sarina Harshuk-Shabso, Flavia Peci, Harry Begthel, Delilah Hendriks, Paul Schürmann, Amanda Andersson-Rolf, Susana M Chuva de Sousa Lopes, Johan H van Es, Ruben van Boxtel, Hans Clevers
来源：
Stem Cell Research & Therapy
摘要：
CRISPR/Cas9介导的基因组工程优化，产生了具有修复突变和疾病建模潜力的碱基编辑酶。在此，我们展示了碱基编辑酶在人体脂肪来源的器官样体内生成复杂肿瘤模型的应用。首先，我们展示了胃肝细胞器官样体中胞嘧啶和腺嘌呤碱基编辑酶在对CTNNB1热点突变建模方面的效果。接下来，我们使用膜内细胞器官样体中的C >T碱基编辑酶插入PTEN的无意义突变，并展示了即使在杂合状态下也表现出肿瘤形成能力。此外，对携带PTEN或PTEN和PIK3CA突变的器官样体进行药物敏感性测定揭示了子宫内膜肿瘤初始阶段的机制。为了进一步扩大基因编辑的范围，我们同时结合SpCas9和SaCas9，在个体靶位点实现C >T和A >G的编辑。最后，我们展示了通过同时转染sgRNA靶向五个癌基因，基因编辑多重复合允许在一步中对结直肠肿瘤形态进行建模。© 2023. Springer Nature Limited.
Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.© 2023. Springer Nature Limited.