T315I mutation of BCR-ABL1 into human Philadelphia chromosome-positive leukemia cell lines by homologous recombination using the CRISPR/Cas9 system

In many cancers, somatic mutations confer tumorigenesis and drug-resistance. The recently established clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a potentially elegant approach to functionally evaluate mutations in cancers. To reproduce mutations by homologous recombination (HR), the HR pathway must be functional, but DNA damage repair is frequently impaired in cancers. Imatinib is a tyrosine kinase inhibitor for BCR-ABL1 in Philadelphia chromosome-positive (Ph+) leukemia, and development of resistance due to kinase domain mutation is an important issue. We attempted to introduce the T315I gatekeeper mutation into three Ph+ myeloid leukemia cell lines with a seemingly functional HR pathway due to resistance to the inhibitor for poly (ADP) ribose polymerase1. Imatinib-resistant sublines were efficiently developed by the CRISPR/Cas9 system after short-term selection with imatinib; resulting sublines acquired the T315I mutation after HR. Thus, the usefulness of CRISPR/Cas9 system for functional analysis of somatic mutations in cancers was demonstrated.


Introduction of T315I mutation in K567.
Among four Ph+ myeloid cell lines, we first used K562, which is the most commonly used Ph+ myeloid cell line for the study of TKI sensitivity. To increase HR efficiency, we treated K562 cells with SCR7 (an inhibitor for a DNA ligase IV that is a key enzyme for NHEJ) 33,34 before and during treatment of the CRISPR/Cas9. We electroporated recombinant Cas9 protein in combination with either forward or reverse sgRNAs and either sense or anti-sense ssODNs. Forty-eight hours after transfection (Fig. 2b), cells were plated into 10 wells of 24-well plate and cultured for 5 more days in the absence of imatinib. Subsequently, the cells were cultured in the presence of 1 μM of imatinib. When the imatinb-resistant cells were selectively expanded, the cells were transferred to culture flask and expanded in the absence of imatinib for further experiments. Imatinib-resistant sublines were obtained in all four combinations of sgRNAs and ssODNs up to day 26 of the imatinib selection (Fig. 2c), while no imatinib-resistant cells were expanded in parental cells during imatinib selection for 30 days (data not shown). Among the four combinations, the combination of SCIenTIfIC RepoRts | (2018) 8:9966 | DOI: 10.1038/s41598-018-27767-6 reverse sgRNA and anti-sense ssODN revealed significantly higher efficiency than the three other combinations (p < 0.001 in Kaplan-Meier test). To verify whether imatinib-resistant sublines acquired the T315I mutation as a result of HR-mediated gene editing, we extracted genomic DNA, amplified the 427 bp fragment of the ABL1 gene containing exon 6 by PCR using primers in introns 5 and 6, and subsequently tested EcoRI digestion of each PCR product (Fig. 2d). PCR products of all seven sublines tested were partially digested with EcoRI, whereas that of parental cells was not. Direct sequencing (Fig. 2e) confirmed mixture of T315I and additional silence point mutations with wild-type sequence in each imatinib-resistant subline, indicating that imatinib-resistant sublines acquired the T315I mutation as a result of HR-mediated gene editing with the CRISPR/Cas9 system. Significance of SCR7 treatment in the HR efficiency. Next, we verified the significance of SCR7 33,34 treatment in HR efficiency. We electroporated recombinant Cas9 protein in combination with reverse sgRNA and anti-sense ssODN (which showed the highest HR efficiency in the above analyses) into K562 cells with or without SCR7 treatment. We also electroporated recombinant Cas9 protein in combination with reverse sgRNA and carrier ssODN (which is not template for HR but enables to induce similar transfection efficiency) into K562 cells treated with SCR7. Electroporated cells were cultured in the same way as shown in Fig. 2b. Imatinib-resistant sublines were obtained relatively faster in the cells treated with SCR7 ( Fig. 2f) (up to day 17 of imatinib selection) than the untreated cells (up to day 22), although no statistically significant difference was observed between the two groups (p = 0.198 in Kaplan-Meier test). Of note, no imatinib-resistant subline was obtained without specific template ssODN during imatinib selection over 30 days.
Induction of T315I mutation in TCCS and KOPM28 using the CRISPR/Cas9 system. Next, we introduced the T315I mutation into two other Ph+ myeloid cell lines (TCCS and KOPM28) resistant to olaparib (Fig. 1). We electroporated recombinant Cas9 protein in combination with reverse sgRNA and anti-sense ssODN into two cell lines treated with SCF7. The electroporated cells were cultured in the same way as shown in Fig. 2b. Imatinib-resistant sublines were efficiently obtained (Fig. 3a) in both TCCS (up to day 9 of imatinib selection) and KOPM28 (up to day 13). Parental cells of TCCS and KOPM28 were completely killed during 14 days culture in the presence of 1 μM imatinib (data not shown). PCR products of genomic DNA from 12 imatinib-resistant sublines of TCCS and KOPM28 were partially digested with EcoRI, unlike those from parental cells (Fig. 3b). Direct sequence of the PCR products from three representative sublines of TCCS (Fig. 3c) and KOPM28 (Fig. 3d) revealed T315I and additional silence point mutations as the main signal, indicating that imatinib-resistant sublines acquired the T315I mutation as a result of HR-mediated gene editing.

No sign of additional mechanisms for imatinib-resistance in T315I-acquired sublines.
Upregulation of BCR-ABL1 protein due to amplification of the BCR-ABL1 fusion gene 18 and overexpression of P-gp 19 were observed in the imatinib-resistant sublines established after long-term culture of imatinib-sensitive Ph+ leukemia cell lines in the presence of increasing concentrations of imatinib. Thus, T315I sublines of three cell lines may acquire these additional mechanisms for imatinib-resistance during imatinib selection. To rule out these possibilities, we evaluated BCR-ABL1 protein expression level in parental cells and imatinib-resistant sublines of three cell lines by Western blotting (Fig. 4a). BCR-ABL1 protein expression levels were similar in both parental cells and imatinib-resistant sublines (T315I #1 and #2) of three cell lines. Thus, amplification of the BCR-ABL1 fusion gene is unlikely to be associated with imatinib-resistant phenotype. Next, we analyzed cell surface expression level of P-gp by flow cytometry (Fig. 4b). P-gp expression in three parental cell lines showed different patterns: K562 showed two peaks of major negative and minor positive populations, while TCCS and KOPM28 showed single negative and single positive population, respectively. The expression patterns of P-gp in imatinib-resistant sublines of three cell lines (T315I #1 and #2) were identical to their parental cells thus demonstrating that upregulation of P-gp was not associated with the imatinib-resistant phenotype.
Acquisition of T315I mutation in ABL1 transcripts of imatinib-resistant sublines. In western blotting analysis (Fig. 4a), ABL1 protein was detectable in K562 but not in TCCS and KOPM28. ABL1 gene consists of 11 exons with alternative splicing exons of 1a and 1b 35 . In the p210 type of BCR-ABL1 fusion, exons 2-11 of the ABL1 gene are fused to exons 1-13 of the BCR gene 35 . Thus, exons 1a and 1b of the ABL1 gene are specific for ABL1 gene, while exons 2-11 of the ABL1 gene are present in both ABL1 and BCR-ABL1 transcripts. Consistent with protein expression, ABL1-specific RT-PCR products with primers in exons 1a or 1b and 2 of the ABL1 gene were detectable in K562 but not in TCCS and KOPM28 (Fig. 5a). In contrast, RT-PCR products with primers in exons 6 and 7 were detectable in three cell lines. These observations demonstrated that K562 has ABL1 and BCR-ABL1 alleles whereas TCCS and KOPM28 have only the BCR-ABL1 allele. Thus, ABL1 allele is another target of the CRISPR/Cas9 system in K562. To distinctively evaluate the ABL1 and BCR-ABL1 alleles in K562 cells, we performed RT-PCR analyses using two sets of primers (Fig. 5b). Primers in exons 1a/b and 6 of  the ABL1 gene were specific for ABL1 transcripts, while those in exon 13 of the BCR gene and exon 6 of the ABL1 gene were specific for BCR-ABL1 transcripts. We directly sequenced the RT-PCR products of parental cells as well as five imatinib-resistant sublines of K562 with the reverse primer (Fig. 5b). BCR-ABL1 transcripts showed two patterns; sublines #1 and #2 revealed mixed pattern of native and HR sequences, while sublines #11, #12, and #13 predominantly showed HR sequence. Taken together, all five sublines had T315I mutation of the BCR-ABL1 transcripts as a result of HR-mediated gene editing. ABL1 transcripts in imatinib-resistant sublines showed diverse patterns (Fig. 5c); subline #1 revealed mixed pattern of native and HR sequences, subline #2 mainly revealed native sequence, subline #11 mainly revealed HR sequence, subline #12 revealed mixed pattern of native sequence and one base deletion due to NHEJ, and subline #13 revealed mixed pattern of one base insertion due to NHEJ and HR sequence. Taken together, T315I mutation due to HR-mediated gene editing was detectable in three of five sublines.
Imatinib-resistance in T315I-acquired sublines. To precisely confirm the effect of the T315I mutation, we first analyzed the phosphorylation status of CRKL (one of critical downstream key enzymes of BCR-ABL1) 36,37 using flow cytometry (Fig. 6a). In the parental cells of three cell lines, CRKL was dephosphorylated by imatinib treatment. In contrast, in the T315I-acquired subline of three cell lines, CRKL was constitutively phosphorylated, even after imatinib treatment. Next, we analyzed the induction of cell cycle arrest (Fig. 6b) and apoptotic cell death (Fig. 6c) by imatinib using flow cytometry. Imatinib treatment of parental cells induced accumulation into the G0/G1 phase in K562 and KOPM28. Furthermore, in parental cells of TCCS, over half of the treated cells accumulated into the sub-G0/G1 phase. In contrast, in T315I-acquired sublines of three cell lines, cell cycle arrest was not induced by imatinib treatment. Furthermore, imatinib treatment induced apoptotic cell death in parental cells, but not in the T315I-acquired sublines of three cell lines. We finally determined dose-response curves of imatinib using the alamarBlue cell viability assay (Fig. 6d). T315I-acquired sublines of three cell lines showed a marked resistance to imatinib (up to 10 μM).

Drug sensitivities of T315I-acquired sublines.
We evaluated the sensitivities to second-generation TKIs using the alamarBlue cell viability assays. T315I-acquired sublines of three cell lines showed marked resistance to nilotinib (Fig. 7a) and dasatinib (Fig. 7b) -up to 1 μM and 100 nM, respectively. Next, we evaluated the sensitivity to ponatinib (Fig. 7c). Parental cells and T315I-acquired sublines of K562 showed similar dose-response curves, and their IC 50 values were approximately 100 nM, which was lower than mean peak concentration (137 nM) of orally administrated ponatinib (45 mg daily) at steady state 38 . Parental cells of TCCS and KOPM28 were highly sensitive to ponatinib and their IC 50 values were less than 0.1 nM. T315I sublines of TCCS and KOPM28 were significantly more resistant to ponatinib than their parental cells; their IC 50 values were approximately 0.2 nM and 20 nM, respectively. We also analyzed the sensitivities to chemotherapeutic agents, AraC and DNR (Fig. 7d) except for K562, which were highly resistant to these agents (data not shown). Parental cells and T315I-acquired sublines of TCCS and KOPM28 were equally sensitive to AraC and DNR, indicating that the T315I mutation of BCR-ABL1 specifically induced resistance to TKI but not to chemotherapeutic agents.

Discussion
In the present study, we tried to introduce the T315I mutation of BCR-ABL1 into Ph+ leukemia cell lines by HR-mediated gene editing with the CRISPR/Cas9 system. Since BCR-ABL1 has been reported to repress a variety of genes involved in the HR pathway 24-28 , we analyzed their sensitivities to olaparib (a PARP1 inhibitor) to functionally evaluate the status of the HR pathway. We found that all four Ph+ myeloid leukemia cell lines were highly resistant, suggesting that their HR pathway is functionally not impaired. Indeed, we successfully established imatinib-resistant sublines of three Ph+ myeloid cell lines-K562, TCCS, and KOPM28-which acquired the T315I mutation as a result of HR-mediated gene editing. We previously tried to introduce specific gene mutations of our interested genes in four Ph− ALL cell lines with the same strategy, but we failed to obtain sublines with the desired mutation. Of note, in the present study, all of the four cell lines that we used in the previous attempts turned out to be sensitive to PARP-1 inhibitor, suggesting that their HR pathway was functionally impaired. Thus, although further verifications are required, sensitivity of cancer cell lines to PARP1 inhibitor may be a useful biomarker to predict efficiency of HR-mediated gene editing with the CRISPR/Cas9 system.
To select successfully integrated cells, we used imatinib. In the previous reports [15][16][17] , imatinib-resistant sublines with T315I were established from Ph+ leukemia cell lines after 5-10 months' culturing in the presence of increasing concentrations of imatinib. Long-term culture in the presence of increasing concentrations of imatinib also induced enhancement of BCR-ABL1 expression 18 and/or upregulation of cell surface expression of P-gp 19 as mechanisms for imatinib-resistance, suggesting that T315I-positive sublines of Ph+ leukemia cell lines may acquire additional mechanism(s) for imatinib-resistance during imatinib selection. Considering these previous reports, to allow an expansion of T315I integrated cells and prevent a spontaneous emergence of imatinib-resistant sublines, we initially cultured cells for 7 days in the absence of imatinib after electroporation. Furthermore, we used 1 μM of imatinib for selection to eliminate emergence of clones that are moderately resistant to imatinib due to mechanism(s) other than an acquisition of T315I mutation. As a result, we successfully expanded T315I acquired sublines of three myeloid Ph+ leukemia cell lines after short-term (7 to 26 days) imatinib selection. Enhancement of BCR-ABL1 expression and upregulation of cell surface expression of P-gp were not induced in imatinib-resistant sublines of three cell lines.
We prepared forward and reverse sgRNAs in combination with sense and anti-sense ssODNs. Efficiency of HR-mediated gene editing is reported to be affected by structure and composition of sgRNA and ssODN such as incorporation of silent mutation to block re-cutting of repaired loci 39 , distance between mutation site and cleavage site 39 , length of homology arms 40,41 , and strand complementarity or orientation of ssODN [40][41][42] . In the present study, all four combinations produced identical distance between mutation site and cleavage site and introduced three silent mutations to block re-cutting of repaired loci. The highest efficiency was observed in the combination of reverse sgRNA and anti-sense ssODN, which was complementary to the transcribed strand and contained the NGG PAM sequence. This was inconsistent with previous reports using different delivery and modality of CRISPR/Cas9 system, since higher efficiency was reported to be obtained by ssODN that was complementary to the non-transcribed strand 42 and by ssODN that was complementary to the PAM strand 41 . Thus, delivery and modality of CRISPR/Cas9 system might affect an optimal combination of sgRNA and ssODN.
TCCS and KOPM28 have the BCR-ABL1 allele but not the ABL1 allele. We confirmed that all of the imatinib-resistant sublines of two cell lines had both native and recombination alleles of BCR-ABL1, suggesting that these two cell lines may serve as good models of acquired T315I mutation in BCR-ABL1. In contrast to TCCS and KOPM28, K562 has both BCR-ABL1 and ABL1 alleles, and its ABL1 allele also acquired the T315I mutation. Although it is unlikely that the induced ABL1 T315I mutation contributes to imatinib resistance, this may be a confounding issue limiting the use of the K562 subclones for drug testing. Removal of the ABL1 allele by a CRISPR/Cas9-induced deletion in K562 may resolve this problem.
T315I sublines from the three cell lines showed a marked resistance to dasatinib and nilotinib, as well as imatinib in comparison with their parental cells. Although ponatinib was developed as a potent TKI that can inhibit all critical kinase domain mutations including T315I, its activity against the T315I mutant was reported to be modestly decreased in comparison with the activity against native ABL1 and BCR-ABL1 as follows 10 . The IC 50 value of ponatinib in the kinase assay in vitro was reported to be 0.37 and 2 nM for native ABL1 and T315I mutant, respectively. In Baf3 transfectants, the IC 50 value of ponatinib was reported to be 0.5 and 11 nM for native BCR-ABL1 and T315I mutant, respectively 10 . In the present study, T315I sublines of TCCS and KOPM28 were sensitive to ponatinib, and their IC 50 values (approximately 0.2 and 20 nM, respectively) were significantly higher than those in parental cells (less than 0.1 nM). In K562, the sensitivities of parental cells and T315I sublines were identical. These observations demonstrated that parental cells and T315I sublines of three cell lines are useful models for the analysis of TKI sensitivities.
Brabetz et al. 43 recently introduced an oncogenic mutation (R140Q) of the IDH2 gene into K562 cells successfully using the CRISPR/Cas9 system. Since R140Q is located in exon 4 of the IDH2 gene, they inserted exons 4-11 with R140Q as a partial cDNA into exon 4 of the IDH2 gene locus via HR. Thus, the endogenous IDH2 gene promoter regulated the integrated IDH2 R140Q mutation, but the insertion lacks introns and 3′-untranslated regions (UTRs) in the target gene. Recently, it has been reported that introns 44 and 3′UTRs 45,46 are involved in transcriptional and post-transcriptional regulation of gene expression. Indeed, 3′UTR of the BCR-ABL1 transcripts were reported to be target of microRNA-203 (miR-203) 47 , miR-424 48 and miR-30e 49 . In the present study, the introduction of a precise mutation into the BCR-ABL1 gene by harnessing the cells' HR repair pathway enabled preservation of other regulatory regions in the gene and minimized off-target effects relative to the more disruptive changes induced by NHEJ driven repair.  Table 1 were Introduction of the T315I mutation by the CRISPR/Cas9 system. Two CRISPR guide sequences were designed using the CRISPR design tool (CRISPR DESIGN, http://crispr.mit.edu) as follow; 5′-atcactgagttcatgacctacgg-3′ (forward sgRNA) and 5′-aactcagtgatgatatagaacgg-3′ (reverse sgRNA). 102 base pair template ssODN in a transcribed strand (sense) direction and its complementary template ssODN in a non-transcribed strand (anti-sense) direction were synthesized by Integrated DNA technologies (Coralville, IA, USA). The sequence of ssODN in the sense direction was as follows; 5′-gggtctgcacccgggagcccccgttctatatcatTaTCgaAttcatgacctacgggaacctcctggactacctgagggagtgcaaccggcaggaggtgaacg-3′. Four mutated nucleotides are capitalized. As a negative control, we used carrier ssODN (Integrated DNA technologies). Recombinant Cas9 nuclease with guide RNA (Integrated DNA technologies) was electroporated into cell lines as a ribonucleoprotein (RNP) complex with template ssODN following manufacturer's protocol 50 . Briefly, RNP complex and template ssODN were electroporated into 1 × 10 5 cells, which were untreated or pretreated with 10 nM of SCR7 33,34 (Cayman Chemical, Ann Arbor, MI, USA) for 24 hours before electroporation, using Neon electroporation transfection system (Thermo Fisher Scientific). The electroporated cells were cultured in the absence or presence of 10 nM of SCR7 for 24 hours in one well of 96 well plate. Forty-eight hours after electroporation, the electroporated cells were mixed with untreated cells and plated into 10-12 wells of 24-well plate. After 5 days expansion in the absence of imatinib, the cells were cultured in the presence of 1 μM of imatinib. During imatinib selection, half of the culture medium in each well was exchanged every 2-3 day with fresh medium containing 1 μM of imatinib. When the imatinb-resistant cells were selectively expanded, imatinb-resistant sublines in each well were transferred to culture flask and expanded in the absence of imatinib for further experiments.

Flow cytometric analyses.
To evaluate cell surface expression of P-glycoprotein, cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-P-glycoprotein antibody (Nichirei, Tokyo, Japan) and analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA). To analyze phosphorylation status of CRKL, cell cycle, and apoptotic cell death, cells were cultured in the presence or absence of 1 μM of imatinib for 24 hours except for detection of apoptosis in K562 cells (60 hours incubation). To evaluate phosphorylation status of CRKL, the cells were fixed with 10% formaldehyde at 37 °C for 10 min and permeabilized with 90% methanol on ice for 30 min. After washing the cells twice with PBS/0.5% BSA, the cells were stained with anti-phospho CRKL (pTyr207) (#3811, Cell Signaling Technology, Danvers, MA, USA) and subsequently with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) (#A11008, Invitrogen, Carlsbad, CA, USA) and analyzed by flow cytometry. For cell cycle analysis, the cells fixed with 70% ethanol were stained with propidium iodide (PI) (Sigma, St. Louis, MO) and analyzed by flow cytometry. To detect apoptosis, the cells were stained with FITC-conjugated Annexin-V and 7AAD (MBL, Nagoya, Japan) and analyzed by flow cytometry.