Selective targeting of the oncogenic KRAS G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression

被引:48
作者
Gao, Qianqian [1 ,2 ,3 ]
Ouyang, Wenjie [1 ,2 ,3 ]
Kang, Bin [1 ,2 ,3 ]
Han, Xu [1 ,2 ,3 ]
Xiong, Ying [4 ]
Ding, Renpeng [1 ,2 ,3 ,5 ]
Li, Yijian [1 ,2 ,3 ]
Wang, Fei [1 ,2 ,3 ,5 ]
Huang, Lei [1 ,2 ,3 ,5 ]
Chen, Lei [1 ,2 ,3 ]
Wang, Dan [1 ,2 ,3 ]
Dong, Xuan [1 ,2 ,3 ]
Zhang, Zhao [1 ,2 ,3 ]
Li, Yanshan [1 ,2 ,3 ]
Ze, Baichen [1 ,2 ,3 ]
Hou, Yong [1 ,2 ,3 ]
Yang, Huanming [1 ,2 ,3 ,6 ]
Ma, Yuanyuan [4 ]
Gu, Ying [1 ,2 ,3 ]
Chao, Cheng-Chi [1 ,2 ,3 ,7 ]
机构
[1] BGI Shenzhen, Guangdong Prov Key Lab Genome Read & Write, Shenzhen 518083, Peoples R China
[2] BGI Shenzhen, China Natl GeneBank, Jinsha Rd, Shenzhen 518120, Peoples R China
[3] BGI Shenzhen, Guangdong Prov Academician Workstn BGI Synthet Ge, Shenzhen 518083, Peoples R China
[4] Peking Univ, Dept Thorac Surg 2, Key Lab Carcinogenesis & Translat Res, Minist Educ Beijing,Canc Hosp & Inst, Beijing, Peoples R China
[5] Univ Chinese Acad Sci, BGI Educ Ctr, Shenzhen 518083, Peoples R China
[6] James D Watson Inst Genome Sci, Hangzhou 310058, Peoples R China
[7] Ab Vis Inc, Milpitas, CA USA
基金
中国国家自然科学基金;
关键词
KRAS mutation; CRISPR/Cas9; dCas9-KRAB; mRNA-regulating; cancer therapy; K-RAS; GENOME; SPECIFICITIES; CRISPR-CAS9;
D O I
10.7150/thno.42325
中图分类号
R-3 [医学研究方法]; R3 [基础医学];
学科分类号
1001 ;
摘要
Rationale: KRAS is one of the most frequently mutated oncogenes in cancers. The protein's picomolar affinity for GTP/GDP and smooth protein structure resulting in the absence of known allosteric regulatory sites makes its genomic-level activating mutations a difficult but attractive target. Methods: Two CRISPR systems, genome-editing CRISPR/SpCas9 and transcription-regulating dCas9-KRAB, were developed to deplete the KRAS G12S mutant allele or repress its transcription, respectively, with the goal of treating KRAS-driven cancers. Results: SpCas9 and dCas9-KRAB systems with a sgRNA targeting the mutant allele blocked the expression of the mutant KRAS gene, leading to an inhibition of cancer cell proliferation. Local adenoviral injections using SpCas9 and dCas9-KRAB systems suppressed tumor growth in vivo. The gene-depletion system (SpCas9) performed more effectively than the transcription-suppressing system (dCas9-KRAB) on tumor inhibition. Application of both Cas9 systems to wild-type KRAS tumors did not affect cell proliferation. Furthermore, through bioinformatic analysis of 31555 SNP mutations of the top 20 cancer driver genes, the data showed that our mutant-specific editing strategy could be extended to a reference list of oncogenic mutations with high editing potentials. This pipeline could be applied to analyze the distribution of PAM sequences and survey the best alternative targets for gene editing. Conclusion: We successfully developed both gene-depletion and transcription-suppressing systems to specifically target an oncogenic KRAS mutant allele that led to significant tumor regression. These findings show the potential of CRISPR-based strategies for the treatment of tumors with driver gene mutations.
引用
收藏
页码:5137 / 5153
页数:17
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