Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth

Wonjoo Kim, Sangeun Lee, Han Sang Kim, Minjung Song, Yong Hoon Cha, Young Hoon Kim, Jeonghong Shin, Eun Seo Lee, Yeonsoo Joo, Jae J. Song, Eun Ju Choi, Jae W. Choi, Jinu Lee, Moonkyung Kang, Jong In Yook, Min Goo Lee, Yeon Soo Kim, Soonmyung Paik, Hyongbum Kim

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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 adenoassociated 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.

Original languageEnglish
Pages (from-to)374-382
Number of pages9
JournalGenome Research
Volume28
Issue number3
DOIs
Publication statusPublished - 2018 Mar

All Science Journal Classification (ASJC) codes

  • Genetics
  • Genetics(clinical)

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  • Cite this

    Kim, W., Lee, S., Kim, H. S., Song, M., Cha, Y. H., Kim, Y. H., Shin, J., Lee, E. S., Joo, Y., Song, J. J., Choi, E. J., Choi, J. W., Lee, J., Kang, M., Yook, J. I., Lee, M. G., Kim, Y. S., Paik, S., & Kim, H. (2018). Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth. Genome Research, 28(3), 374-382. https://doi.org/10.1101/gr.223891.117