RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.
Genome Med. 2019 Apr 16;11(1):21. doi: 10.1186/s13073-019-0627-9.
CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3'-UTR of mouse β-actin, allowing co-expression of reporter genes or oncogenes from the β-actin promoter. We showed that knock-in of oncogenic Ras and loss of p53 efficiently induced intrahepatic cholangiocarcinoma in mice. Further, our strategy can generate bioluminescent liver cancer to facilitate tumor imaging. This method simplifies in vivo gain-of-function genetics by facilitating targeted integration of oncogenes.
CRISPR/Cas9 技术彻底改变了癌症小鼠模型。虽然 CRISPR/Cas9 的功能丧失遗传学已经得到很好的建立，但由于基因敲入效率低，在体癌细胞模型中产生功能获得性等位基因仍然具有挑战性。在这里，我们开发了基于 CRISPR 的体细胞致癌基因敲入用于癌症建模（CRISPR-SONIC），这是一种使用非同源性修复快速进行体内癌症建模的方法，可将致癌基因整合到靶向基因组位点。我们使用双向导 RNA 策略，将质粒供体整合到小鼠 β-肌动蛋白的 3'UTR 中，允许报告基因或来自 β-肌动蛋白启动子的致癌基因共表达。我们表明，致癌性 Ras 的敲入和 p53 的缺失有效地诱导了小鼠肝内胆管癌。此外，我们的策略可以产生生物发光肝癌，以促进肿瘤成像。该方法通过促进致癌基因的靶向整合，简化了体内功能获得性遗传学。
