Efficient Disruption Of Bcr-abl Gene By CRISPR RNA-guided FokI Nucleases Depresses The Oncogenesis Of Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia(CML)is a myeloproliferative neoplasm initiated from hematopoietic stem cells.It is characterized by t(9;22)(q34;q11)reciprocal translocation that forms a bcr-abl fusion gene which encodes a BCR-ABL protein with constitutive tyrosine kinase activity.The BCR-ABL oncoproteinactivates multiple signaling pathways such as STAT5,MEK-ERK,and CRKL,contributing to the induction of malignant proliferation and apoptosis inhibition.The tyrosine kinase inhibitors(TKIs)are used for the treatment of CML and have achieved a favorable therapeutic effect.However,parts of patients have developed drug resistance to TKIs due to the advent of BCR-ABL kinase domain mutations which affect the binding and activity of TKIs.In addition,the leukemia stem cells(LSCs)of CML lack sensitivity to TKIs,which become an important reason for disease relapse of CML.Therefore,novel therapeutic strategies for CML need to be explored.The CRISPR RNA-guided Fok I nucleases(RFNs)provide us a new therapeutic potential to obliterate this disease.The RFNs adopted in our research meet the merits of variable target sites and specificity of cleavage enabled its suitability for gene editing of CML cells.In our study,we designed several pairs of g RNA with different spacers and then chose the most efficient pair of g RNA to target bcr-abl and recruit dimeric Fok I-d Cas9 to cleave bcr-abl.Meanwhile,exogenous DNA template(donor)containing 8-base of Not I enzyme digestion site was provided to initiate HDR at the DSBs.If the 8-base sequence of Not I enzyme digestion site inserts into the bcr-abl sequence by HDR,the reading frame of bcr-abl will shift and a stop codon may generate ahead of time downstream the RFNs cleavage site,then BCR-ABL translation would prematurely terminate.We studied the ability of the RFNs system to disrupt the bcr-abl oncogene,and evaluated whether it could down-regulate the expression level of BCR-ABL.Then,we detected its effect on imatinib sensitive and resistant CML cell lines and stem/progenitor cells in vitro,and finally explored its effect in xenograft mice models of CML.Methods:(1)Design and construction of g RNA and the disruption of RFNs on bcr-abl gene.The sequence of bcr-abl gene was analyzed in Zi Fi T website to design guide RNA(g RNA).The g RNA expression plasmids were constructed by molecular cloning technique.Donors with homologous arms were constructed according to the target sites,and 8 base of Not I sequence were inserted into donors.After the RFNs and donor were cotransfected into K562 cells,the disruption effect of RFNs on bcr-abl was detected via immunofluorescence assay,T7 endonuclease 1(T7E1)assay,Not I restriction enzyme digestion and Sanger sequencing.K562 and K562/G01 cells were co-delivered with RFNs plus donor,and the expression of BCR-ABL and its downstream signaling molecules were determined by western blotting.(2)Effect and mechanism of RFNs on proliferation and apoptosis of CML cells.To test the effect of RFNs system on proliferation and apoptosis of CML cells,the RFNs were transfected into imatinib sensitive and resistant cells,bcr-abl positive CD34~+ cells and bcr-abl negative cells.Cell viability was detected via colony-forming assay and CCK-8 assay.The effects of RFNs on cell apoptosis were evaluated by DAPI staining and flow cytometry.The activation of PARP and caspase 3 were detected by western blot.(3)Murine xenograft model was adopted to evaluate the capacity of RFNs in attenuating the tumorigenic ability of bcr-abl.To detect whether disruption of bcr-abl could impair its pathogenicity in vivo,the NOD/SCID mice receiving sublethal dose of radiation were intravenously injected with wild type K562/G01 cells or K562/G01 cells cotransfected with g RNA-17 plus donor,RFNs-half plus donor,RFNs plus donor,respectively.The WBC counts,proportion of CD45+ cells,survival time were recorded to evaluate the progression of leukemia.Bone marrow morphology test,histopathologic examination Infiltration and immunofluorescent assay of BCR-ABL protein were performed to evaluate the infiltration of leukemia cells.Results and conclusions:(1)The g RNA expression plasmid targeting bcr-abl was successfully constructed.The results of western blot showed that when g RNA-17,Fok I-d Cas9 and donor were co-delivered into K562 cells,the continuity of bcr-abl gene could be effectively disrupted,thus generated the double strand breaks.The 8-base sequence of Not I was successfully inserted into bcr-abl when codeliver RFNs plus donor into K562.Moreover,the frame shifts mutation of bcr-abl induced by Not I sequence insertion produced a premature stop codon at the downstream of cutting site and fundamentally terminated the bcr-abl translation.Correspondingly,the activation of signal molecules downstream of BCR-ABL was inhibited.(2)Cotransfect RFNs plus donor could suppress proliferation and induce apoptosis of imatinib sensitive and resistant CML cells.And similar result was verified in CML stem/progenitor cells.In contrast,RFNs havehardly any effect on the proliferation and apoptosis of U937,HL60,AD293 cells and bcr-abl negative CD34~+ cells(3)The NOD-SCID mice treated with RFNs plus donor developed better physical condition,less counts of WBC,lower level proportion of CD45+ cells,lighter weight of spleen and liver,gentler infiltration of tissues and longer survival time.These result revealed that the RFNs impaired the leukemogenesis capacity of bcr-abl in mice.In summary,we have shown that the RFNs can function as heterodimer to efficiently edit bcr-abl with high targetability and flexibility.The 8-base sequence of Not I was successfully inserted into the bcr-abl sequence by HDR,thus led to the frame shift of bcr-abl shift and generated a stop codon at the downstream RFNs cleavage site,which prematurely terminated BCR-ABL translation.This approach provides a novel therapeutic option for CML patients affiliated by TKIs resistance or disease relapse.