1. Academic Validation
  2. Efficient disruption of bcr-abl gene by CRISPR RNA-guided FokI nucleases depresses the oncogenesis of chronic myeloid leukemia cells

Efficient disruption of bcr-abl gene by CRISPR RNA-guided FokI nucleases depresses the oncogenesis of chronic myeloid leukemia cells

  • J Exp Clin Cancer Res. 2019 May 28;38(1):224. doi: 10.1186/s13046-019-1229-5.
Zhenhong Luo 1 Miao Gao 2 Ningshu Huang 3 Xin Wang 4 Zesong Yang 4 Hao Yang 1 Zhenglan Huang 5 Wenli Feng 6
Affiliations

Affiliations

  • 1 Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, China.
  • 2 Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China.
  • 3 Department of Clinical Laboratory, The Children's Hospital of Chongqing Medical University, Chongqing, 400016, China.
  • 4 Department of Hematology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China.
  • 5 Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, China. zhenglan@cqmu.edu.cn.
  • 6 Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, China. fengwl@cqmu.edu.cn.
Abstract

Background: The Bcr-Abl fusion gene encodes Bcr-Abl oncoprotein and plays a crucial role in the leukemogenesis of chronic myeloid leukemia (CML). Current therapeutic methods have limited treatment effect on CML patients with drug resistance or disease relapse. Therefore, novel therapeutic strategy for CML is essential to be explored and the CRISPR RNA-guided FokI nucleases (RFNs) meet the merits of variable target sites and specificity of cleavage enabled its suitability for gene editing of CML. The RFNs provide us a new therapeutic direction to obliterate this disease.

Methods: Guide RNA (gRNA) expression plasmids were constructed by molecular cloning technique. The modification rate of RFNs on Bcr-Abl was detected via NotI restriction Enzyme digestion and T7 Endonuclease 1 (T7E1) assay. The expression of Bcr-Abl and its downstream signaling molecules were determined by western blotting. The effects of RFNs on cell proliferation and Apoptosis of CML cell lines and CML stem/progenitor cells were evaluated by CCK-8 assay and flow cytometry. In addition, murine xenograft model was adopted to evaluate the capacity of RFNs in attenuating the tumorigenic ability of Bcr-Abl.

Results: The RFNs efficiently disrupted Bcr-Abl and prematurely terminated its translation. The destruction of Bcr-Abl gene suppressed cell proliferation and induced cell Apoptosis in CML lines and in CML stem/progenitor cells. Moreover, the RFNs significantly impaired the leukemogenic capacity of CML cells in xenograft model.

Conclusion: These results illustrate that the RFNs can target to disrupt Bcr-Abl gene and may provide a new therapeutic option for CML patients affiliated by drug resistance or disease relapse.

Keywords

Bcr-abl; Chronic myeloid leukemia; Homology-directed repair; Leukemogenesis; RNA guided-FokI nucleases.

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