KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.

KRAS是人类肿瘤中最常突变的癌基因，其激活突变是重要的治疗靶点。 Cas9 与 CRISPR-Cas 系统中的引导 RNA 相结合，可识别特定的 DNA 序列并产生双链断裂，从而能够编辑相关基因。在这里，我们利用 CRISPR 特异性靶向癌细胞中的突变KRAS等位基因。我们使用报告系统筛选向导RNA，并在慢病毒递送Cas9和向导RNA后在癌细胞中验证它们。 Cas9 和向导 RNA 递送后，测定体外癌细胞的存活、增殖和致瘤性以及体内肿瘤的生长。我们鉴定出能够有效靶向突变型KRAS的引导 RNA，而不会对野生型等位基因造成显着改变。在用编码 Cas9 的慢病毒载体转导的KRAS突变癌细胞中，多西环素诱导表达该指导 RNA，破坏了突变KRAS基因，从而在体外和体内抑制癌细胞增殖。在免疫缺陷小鼠中，肿瘤内注射表达 Cas9 和 sgRNA 的慢病毒和腺相关病毒可抑制体内肿瘤生长，尽管不完全。含有野生型KRAS的细胞中 Cas9 和指导 RNA 的表达在体外和体内均不会改变细胞存活或增殖。我们的研究提供了一个概念证明，即 CRISPR 可用于在体外和体内靶向癌症的驱动突变。