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

7 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

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Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Growth
Neoplasms
CRISPR-Cas Systems
Alleles
Cell Proliferation
Lentivirus
Mutation
Doxycycline
Oncogenes
Cell Survival

All Science Journal Classification (ASJC) codes

  • Genetics
  • Genetics(clinical)

Cite this

Kim, W., Lee, S., Kim, H. S., Song, M., Cha, Y. H., Kim, Y. H., ... 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
Kim, Wonjoo ; Lee, Sangeun ; Kim, Han Sang ; Song, Minjung ; Cha, Yong Hoon ; Kim, Young Hoon ; Shin, Jeonghong ; Lee, Eun Seo ; Joo, Yeonsoo ; Song, Jae J. ; Choi, Eun Ju ; Choi, Jae W. ; Lee, Jinu ; Kang, Moonkyung ; Yook, Jong In ; Lee, Min Goo ; Kim, Yeon Soo ; Paik, Soonmyung ; Kim, Hyongbum. / Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth. In: Genome Research. 2018 ; Vol. 28, No. 3. pp. 374-382.
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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.",
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Kim, W, Lee, S, Kim, HS, Song, M, Cha, YH, Kim, YH, Shin, J, Lee, ES, Joo, Y, Song, JJ, Choi, EJ, Choi, JW, Lee, J, Kang, M, Yook, JI, Lee, MG, Kim, YS, Paik, S & Kim, H 2018, 'Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth', Genome Research, vol. 28, no. 3, pp. 374-382. https://doi.org/10.1101/gr.223891.117

Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth. / Kim, Wonjoo; Lee, Sangeun; Kim, Han Sang; Song, Minjung; Cha, Yong Hoon; Kim, Young Hoon; Shin, Jeonghong; Lee, Eun Seo; Joo, Yeonsoo; Song, Jae J.; Choi, Eun Ju; Choi, Jae W.; Lee, Jinu; Kang, Moonkyung; Yook, Jong In; Lee, Min Goo; Kim, Yeon Soo; Paik, Soonmyung; Kim, Hyongbum.

In: Genome Research, Vol. 28, No. 3, 03.2018, p. 374-382.

Research output: Contribution to journalArticle

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AU - Kim, Wonjoo

AU - Lee, Sangeun

AU - Kim, Han Sang

AU - Song, Minjung

AU - Cha, Yong Hoon

AU - Kim, Young Hoon

AU - Shin, Jeonghong

AU - Lee, Eun Seo

AU - Joo, Yeonsoo

AU - Song, Jae J.

AU - Choi, Eun Ju

AU - Choi, Jae W.

AU - Lee, Jinu

AU - Kang, Moonkyung

AU - Yook, Jong In

AU - Lee, Min Goo

AU - Kim, Yeon Soo

AU - Paik, Soonmyung

AU - Kim, Hyongbum

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N2 - 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.

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Kim W, Lee S, Kim HS, Song M, Cha YH, Kim YH et al. Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth. Genome Research. 2018 Mar;28(3):374-382. https://doi.org/10.1101/gr.223891.117