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 language | English |
|---|---|
| Pages (from-to) | 374-382 |
| Number of pages | 9 |
| Journal | Genome Research |
| Volume | 28 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2018 Mar |
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All Science Journal Classification (ASJC) codes
- Genetics
- Genetics(clinical)
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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 journal › Article
TY - JOUR
T1 - Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth
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
PY - 2018/3
Y1 - 2018/3
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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85046083420&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85046083420&partnerID=8YFLogxK
U2 - 10.1101/gr.223891.117
DO - 10.1101/gr.223891.117
M3 - Article
AN - SCOPUS:85046083420
VL - 28
SP - 374
EP - 382
JO - Genome Research
JF - Genome Research
SN - 1088-9051
IS - 3
ER -