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

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.

Fig. 1

Schematic diagram of dimeric RFNs target bcr-abl to induce gene modification. a Dimeric FokI-dCas9 proteins target to DNA half-site sequences with PAM out orientation by pairs of gRNAs to cleave the intervening spacer sequence of c-abl exon 2. The gRNA-15, gRNA-16, gRNA-17, and gRNA-half showed the target site in c-abl exon 2. b The structure of FokI-dCas9 nucleases expression plasmid

Fig. 2

RFNs induce gene modification of bcr-abl. a Detection of BCR-ABL expression by western blot. Vehicle or each gRNA combined FokI-dCas9 plus donor were co-transfected into K562 cells. b Examination of the amount of γH2AX by western blot. K562 cells were transfected with vehicle or each gRNA guided FokI-dCas9 plus donor. c DSBs induced by RFNs were detected with 53BP1 immunostaining. Etoposide treated K562 cells were adopted as positive control and wild type K562 cells were used as negative control. Different groups of plasmids transfected K562 cells were harvested after 60 h of treatment. Foci that form more than 3 in cells were considered to be 53BP1 positive and the positive rates were shown beneath each panel. d The HDR rate of RFNs plus donor on bcr-abl was detected by NotI digestion. The “uncut” indicated the location of pcr fragment of wild type and “cut” showed the location of digested fragment by NotI restriction enzyme. e Predicted HDR was verified by Sanger sequencing. The sequence of RFNs plus donor treated cells was analyzed with in silico analysis. Result showed the NotI sequence was inserted into bcr-abl and a TGA stop codon was generated downstream of the cleavage site. f The edited rate by RFNs was detected via T7E1 assay. The “cut” bands indicated occur of “indels”

Fig. 3

The expressions of BCR-ABL oncoprotein and its downstream signaling molecules were decreased in RFNs treated CML cells. K562 and K562/G01 cells were co-delivered with gRNA-17 plus donor, RFNs-half plus donor, RFNs plus donor, respectively. Proteins were extracted after 60 h of transfection and analyzed by western blot. Expressions of p-BCR-ABL and BCR-ABL were reduced in RFNs plus donor group (a). The activated phospho-CRKL, phospho-ERK and phospho-STAT5 were down-regulated in RFNs plus donor group (b)

Fig. 4

RFNs suppress viability and induce apoptosis of imatinib sensitive and resistant cells. Cells were transfected with gRNA-17 plus donor, RFNs-half plus donor, RFNs plus donor, respectively. a Evaluation of cell proliferative capacity by CCK-8 assay. b, c The capacity of colony formation was assessed by colony-forming assay. d Cell nucleuses were stained by DAPI to detect the morphologic changes caused by apoptosis. The white arrows pointed out the typical apoptotic cells. e Satistical analysis result of apoptotic cells tested by flow cytometry. f The activation of apoptotic pathway was investigated by western blot. The results are presented as the means ± SD. One-way ANOVA analysis was used to compare treated groups with control group, p < 0.01 (**) and p < 0.001 (***)

Fig. 5

The proliferation was suppressed and apoptosis was induced by RFNs in CML stem/progenitor cells. CML CD34⁺ cells were transfected with gRNA-17 plus donor, and RFNs plus donor. a The evaluation of modification efficiency of RFNs on bcr-abl by NotI digestion. b Cell viability of CML stem/progenitor cells was tested by CCK-8 assay. c, d Percentage of apoptotic CML stem/progenitor cells was tested by flow cytometry. All results are exhibited as the mean ± SD, p < 0 .01 (**) and p < 0.001 (***)

Fig. 6

The leukemogenic capacity of bcr-abl in mice was impaired by RFNs. a WBC counts in each group were counted. The maximum value of each mouse was recorded. b, c The weights of spleen and liver of mice in each group were measured. d The amount of human CD45⁺ cells was tested by flow cytometry. e The expression of BCR-ABL protein in each group was detected by immunofluorescent assay. f Immature cells from bone marrow were checked by Wright’s stain and the infiltration of spleen and liver was analyzed by H&E stain. g Kaplan-Meier method to analyze the survival curves of mice in each group