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

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          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.

          Electronic supplementary material

          The online version of this article (10.1186/s13046-019-1229-5) contains supplementary material, which is available to authorized users.

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          Most cited references 47

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          Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.

          Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity. Copyright © 2013 Elsevier Inc. All rights reserved.
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            High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

            CRISPR RNA-guided endonucleases (RGENs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGENs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We readily detected off-target alterations induced by four out of six RGENs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbor up to five mismatches and many are mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGENs are highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.
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              Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia.

              The cause of chronic myeloid leukemia (CML) is a constitutively active BCR-ABL tyrosine kinase. Imatinib inhibits this kinase, and in a short-term study was superior to interferon alfa plus cytarabine for newly diagnosed CML in the chronic phase. For 5 years, we followed patients with CML who received imatinib as initial therapy. We randomly assigned 553 patients to receive imatinib and 553 to receive interferon alfa plus cytarabine and then evaluated them for overall and event-free survival; progression to accelerated-phase CML or blast crisis; hematologic, cytogenetic, and molecular responses; and adverse events. The median follow-up was 60 months. Kaplan-Meier estimates of cumulative best rates of complete cytogenetic response among patients receiving imatinib were 69% by 12 months and 87% by 60 months. An estimated 7% of patients progressed to accelerated-phase CML or blast crisis, and the estimated overall survival of patients who received imatinib as initial therapy was 89% at 60 months. Patients who had a complete cytogenetic response or in whom levels of BCR-ABL transcripts had fallen by at least 3 log had a significantly lower risk of disease progression than did patients without a complete cytogenetic response (P<0.001). Grade 3 or 4 adverse events diminished over time, and there was no clinically significant change in the profile of adverse events. After 5 years of follow-up, continuous treatment of chronic-phase CML with imatinib as initial therapy was found to induce durable responses in a high proportion of patients. (ClinicalTrials.gov number, NCT00006343 [ClinicalTrials.gov].) Copyright 2006 Massachusetts Medical Society.
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                Author and article information

                Contributors
                369699973@qq.com
                gaomiao_1@163.com
                1026764319@qq.com
                925170788@qq.com
                joycelnn@163.com
                chrisyang1992@163.com
                +8615823826839 , zhenglan@cqmu.edu.cn
                +8613983802837 , fengwl@cqmu.edu.cn
                Journal
                J Exp Clin Cancer Res
                J. Exp. Clin. Cancer Res
                Journal of Experimental & Clinical Cancer Research : CR
                BioMed Central (London )
                0392-9078
                1756-9966
                28 May 2019
                28 May 2019
                2019
                : 38
                Affiliations
                [1 ]ISNI 0000 0000 8653 0555, GRID grid.203458.8, 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 ]GRID grid.452206.7, Department of Laboratory Medicine, , The First Affiliated Hospital of Chongqing Medical University, ; Chongqing, 400016 China
                [3 ]ISNI 0000 0000 8653 0555, GRID grid.203458.8, Department of Clinical Laboratory, , The Children’s Hospital of Chongqing Medical University, ; Chongqing, 400016 China
                [4 ]GRID grid.452206.7, Department of Hematology, , The First Affiliated Hospital of Chongqing Medical University, ; Chongqing, 400016 China
                Article
                1229
                10.1186/s13046-019-1229-5
                6537404
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: No.81500129
                Award ID: No.81572060
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2019

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