ARTICLE https://doi.org/10.1038/s41467-020-18875-x OPEN In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells M. Martinez-Lage 1,10, R. Torres-Ruiz 1,2,10 ✉, P. Puig-Serra 1, P. Moreno-Gaona1, M. C. Martin1, F. J. Moya1, O. Quintana-Bustamante 3,4, S. Garcia-Silva 5, A. M. Carcaboso 6,7, P. Petazzi2, C. Bueno2, J. Mora 6,7, H. Peinado 5, J. C. Segovia 3,4, P. Menendez2,8,9 & S. Rodriguez-Perales 1 ✉ 1234567890():,; Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells. 1 Molecular Cytogenetics and Genome Editing Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas (CNIO), 28029 Madrid, Spain. 2 Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, 08036 Barcelona, Spain. 3 Differentiation and Cytometry Unit, Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), 28040 Madrid, Spain. 4 Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), 28040 Madrid, Spain. 5 Microenvironment and Metastasis Group, Molecular Oncology Program, Spanish National Cancer Research Centre, 28029 Madrid, Spain. 6 Institut de Recerca Sant Joan de Deu, Barcelona, Spain. 7 Department of Pediatric Hematology and Oncology, Hospital Sant Joan de Deu, 08950 Barcelona, Spain. 8 Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluis Companys, 08010 Barcelona, Spain. 9 Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC), ISCIII, Barcelona, Spain. 10These authors contributed equally: M. Martinez-Lage and R. Torres-Ruiz. ✉email: rtorresr@cnio.es; srodriguezp@cnio.es NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x D espite advances in treatment, cancer continues to cause CRISPR tool development limits the utility of this strategy for over nine million deaths worldwide each year. Indeed, developing targeted cancer therapies. there is still much to be learned about the biologic In the present study, we report an efficient NHEJ CRISPR- behavior of cancer cells and molecular disease mechanisms. In mediated genome editing strategy for targeting FOs, which we this sense, genome editing offers unprecedented opportunities to believe represents a valid approach for directed elimination of further our knowledge of cancer biology and to fuel the devel- cancer cells harboring a given FO. The approach is based on opment of new preclinical models and more efficient and directed targeting two intronic sequences—one in each of the genes strategies to eliminate cancer cells1. Unlike other genetic diseases involved in the FO—that induces a cancer cell-specific genome such as Duchenne muscular dystrophy or cystic fibrosis, cancer deletion that eliminates key protein domains or changes the development involves several genetic mutations that can dereg- reading frame of the FO. Notably, this gene editing-based ulate multiple genes. In the context of cancer gene therapy, it is approach induces deletion only in cells harboring a FO without clear that targeting a single gene is often insufficient to eliminate affecting exonic sequences or protein expression of the germline cancer cells—yet, many types of cancers are addicted to the non-rearranged alleles. The same two single guide RNAs presence of a single oncogenic event that can reprogram cells by (sgRNAs) allow the targeting of different isoforms or every deregulating downstream molecular and (epi)-genetic programs2 patient-specific breakpoint of a given FO, and is thus a universal and initiate tumorigenesis. This is the case for the so-called fusion approach for cancer-associated FOs. In vitro analysis and patient- oncogenes (FOs), which are chimeric genes resulting from in- derived xenograft (PDX) models show that delivery of CRISPR/ frame fusions of the coding sequences of two genes involved in a Cas9 components targeting a FO results in efficient and specific chromosomal rearrangement3. While the nature of the FOs may tumor growth control. Our findings provide a proof-of-concept be diverse, they are primarily classified as involving transcription for a highly efficient and non-breakpoint-specific genome editing factors or tyrosine kinases. Silencing of FO transcripts has been strategy-targeting FOs as an innovative approach for selective shown to inhibit tumor cell growth in vitro and in vivo4,5, elimination of cancer cells. demonstrating FO addiction in many human cancers. Moreover, genome-wide mutational studies—particularly in childhood cancers—have provided additional support for some FOs as the Results sole necessary genetic drivers for cancer initiation by revealing CRISPR-mediated intronic targeting enables FO disruption. silent mutational landscapes in FO-initiated tumors6,7. Our rationale was to devise a genome editing approach to speci- FOs are recurrent genomic findings in human cancer and are fically disrupt FOs in cancer cells that fulfils two strict criteria: i) it characterized by patient-specific genomic breakpoints that would not affect the exonic sequences or the expression of wild- occur in intronic regions, rarely disrupting coding sequences. type alleles involved in the rearrangement, and ii) it would be Analysis of data from The Cancer Genome Atlas suggests that feasible irrespective of the FO isoform or the patient-specific FOs drive the development of more than 16% of human can- breakpoint. To test this approach, we first used a cellular model of cers8, including mesodermal cancers (typically leukemias, Ewing sarcoma, one of the most common cancers in children/ lymphomas and sarcomas). FOs have also been found in epi- adolescents characterized by a chromosomal translocation that thelial cancers, including prostate9, colorectal10, breast11 or fuses the transactivation domain of the RNA-binding protein melanoma12, and over 350 recurrent FOs involving >300 dif- EWSR1 to the DNA-binding domain of an ETS protein, most ferent genes have been identified to date13,14. Given their commonly FLI1. The EWSR1-FLI1 (EF) fusion acts as a dominant restriction to cancer cells, FOs are attractive targets for directed transcription factor, and cells are addicted to EF expression27–29. therapy; however, therapeutic targeting of specific FOs has Two main EF isoforms exist that fuse exon 7 of EWSR1 to either remained challenging due to the difficulties in specifically exon 5 (EF type 2) or exon 6 of FLI1 (EF type 1)30–32. recognizing and targeting the resultant chimeric protein, and We designed a strategy to induce EF-specific genomic deletion also because FO products are intracellular, necessitating effec- targeting two genomic introns—one in each rearranged gene – tive intracellular approaches for delivery of therapeutic mole- flanking the breakpoint introns. To target all isoforms of EF, four cules targeting the chimeric transcripts/proteins15. Along this sgRNAs were designed to target introns 3 and 6 of EWSR1 and line, small molecules16, intrabodies17 and aptamers18 have been introns 6 and 8 of FLI1 (Fig. 1a and Supplementary Table 1). used successfully to target fusion proteins, and antisense sgRNAs were designed specifically to not disrupt described splice RNA19, ribozymes20 and RNAi21 to target the fusion tran- acceptor or donor sites or transcription regulators such as scripts. Likewise, the development of genome editing approa- enhancers or silencers. We also confirmed that the sgRNA target ches offers new possibilities to directly target and modify the sites were not affected by common single nucleotide polymorph- genomic sequence of cancer cells22. isms (SNPs) (Supplementary Fig. 1a, b). Targeted introns were Clustered Regularly Interspaced Short Palindromic Repeats selected to generate large deletions including key functional (CRISPR)/Cas9-based technology has revolutionized genome domains of the FO, to induce a frameshift event in the remaining editing of mammalian cells, and can generate targeted breaks at 3′ region of the FLI1 gene, and to cover all the common hotspot any desired location in the genome—opening new horizons for introns within the break cluster regions. Consequently, genomic therapeutic gene editing to correct monogenic or somatic deletions will occur only in cells harboring the FO with both on- mutations23,24. CRISPR/Cas9 induced breaks can be repaired by target intronic regions in the same chromosome. Crucially, one of two major double-strand break (DSB) repair pathways: the intron-directed sgRNAs guarantee the germline configuration of inefficient but error-free homology-directed repair (HDR) path- non-rearranged EWSR1 and FLI1 alleles, such that the expression way, which requires a DNA template; and the highly efficient but of wild-type alleles is preserved in healthy cells. error-prone non-homologous end joining (NHEJ) pathway, Using a single sgRNA lentiviral expression vector (pLV- which does not require a DNA template. A recent pioneering U6sgRNA-EFSCas9)33, we tested the efficiency of genomic study by Chen et al. exploited the HDR repair pathway to insert a deletion with four combinations of sgRNAs (sgE3-sgF6, sgE3- suicide gene at patient-specific breakpoints of FOs in prostate and sgF8, sgE6-sgF6, sgE6-sgF8) in the A673 Ewing sarcoma cell line. hepatocellular cancer cell lines25. Although very elegant, the Sanger sequencing analysis of PCR products using oligonucleo- combination of an extremely low efficiency (1–10%) HDR-based tides flanking the targeted loci confirmed genomic deletions approach26 and the requirement of patient-specific breakpoint (Supplementary Fig. 2a), and EF-targeted A673 cells showed a 2 NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x ARTICLE a b U6 H1 EFS Cas9 eGFP Wpre EWSR1-FLI1 LTR LTR sgE3 sgF8 P2A Type 1 sgRNAs sgE3 sgE6 sgF6 sgF8 Type 2 LVCas9_EF DNA RNA Protein Exons 1 2 3 4 5 6 7 5 6 7 8 9 Analysis Ewing sarcoma Ewing sarcoma cell transduced cell Gene EWSR1 FLI1 c d e A673 LVCas9_EF A673 LVCas9_NT A673 LVCas9_EF A673 LVCas9_NT A673 LVCas9_NT A673 LVCas9_EF bp M 2d 4d 6d 2d 4d 6d bp M 2d 4d 6d 2d 4d 6d 3d 6d 10d 3d 6d 10d 1000 1000 wt αFLI1 500 500 (51KDa) 300 Deletion 300 200 Deletion 100 αGAPDH 200 200 (37KDa) ALBUMIN GAPDH 100 100 EWSR1 intron 3 FLI1 intron 8 EWSR1 exon 3 FLI1 exon 9 Fig. 1 Strategy and in vitro CRISPR-mediated disruption of EWSR1-FLI1. a Scheme representing type 1 and 2 EWSR1-FLI1 FOs, illustrating the genomic structure with exon arrangement and sites of fusion. sgRNAs targeting introns 3 and 6 of EWSR1 and 6 and 8 of FLI1 are indicated. b Schematic representation of the all-in-one lentiviral vector for simultaneous expression of two sgRNAs, Cas9 and eGFP regulated by the U6, H1, and EFS promoters. c, Genomic PCR analysis of edited and control A673 cells using oligonucleotides flanking the DNA targeted by sgE3 and sgF8 (n = 3, independent studies). The 300 bp PCR product denotes deletion of the DNA fragment between the loci targeted by sgE3 and sgF8. PCR analysis was performed on extracted DNA of cells at day 2, 4, and 6 post-transduction (pt). ALBUMIN was used as an internal control of the PCR reaction. Bottom panel shows a representative Sanger sequencing chromatogram of the PCR products. d RT-PCR products from edited and control A673 Ewing sarcoma cells (n = 3, independent studies). Analysis was done using extracted RNA of cells at day 2, 4, and 6 pt. Arrows depict the sizes of wild-type (961 bp) and deleted (150 bp) RT-PCR products. GAPDH was used as an internal control of the RT-PCR reaction. Bottom panel shows a representative Sanger sequencing chromatogram of RT- PCR products. e Western blotting of EWSR1-FLI1 in A673 cells. Analysis was done using total protein extracted from cells at day 3, 6, and 10 post- transfection using an antibody specific for FLI1. GAPDH was used as an internal control of the assay. LTR: Long term repeat; P2A: porcine teschovirus-1 2A self-cleaving peptide; WPRE: Virus (WHP) posttranscriptional regulatory elements. significantly blunted clonogenic capacity (51%, 62%, 49%, and (Supplementary Table 1) revealed a PCR band verified by Sanger 56%, respectively) irrespective of the sgRNA pair used (Supple- sequencing both in A673 cells (Fig. 1c) and in RD-ES cells mentary Fig. 2b). Cleavage with sgE3-sgF8 resulted in high (Supplementary Fig. 2c). Consistently, RT-PCR and western blot deletion efficiency, generating the largest (27.7 kb) EF deletion analysis confirmed that the simultaneous expression of sgE3 and and resulting in the complete loss of the EWSR1 transactivation sgF8 induced a robust loss of EF mRNA and protein, respectively domain and a frameshift alteration of the entire FLI1 DNA- (Fig. 1d, e and Supplementary Fig. 2d). We designed a quanti- binding region (Supplementary Fig. 3a). Accordingly, this tative qRT-PCR-based expression assay for FO to estimate rates combination was chosen for further study. Notably, targeted of deletion, and found a significant 4.5-fold decrease in FO deep-sequencing of sgE3- or sgF8-targeted A673 cells revealed expression in edited A673 cells at day 3 post-transduction (p.t.) 62% and 66% insertion/deletion (indels) in EWSR1 and FLI1 on- (Supplementary Fig. 2e). Deep sequencing of the deletion PCR target sites, respectively (Supplementary Table 2a, b). For amplicons indicated a preference (~60%) for precise ligation of subsequent targeting of EF, sgE3 and sgF8 were cloned into an cut products (Supplementary Fig. 2f). Finally, targeted deep- all-in-one expression plasmid34 (pLV-U6sgE3-H1sgF8-EFSCas9- sequencing analysis revealed that EF-targeted human primary 2A-eGFP; hereafter termed LVCas9_EF) expressing sgE3 and mesenchymal stem cells (hMSC) cells perform accurate splicing sgF8 from the U6 and H1 RNApol III promoters, respectively, of EWSR1 exons. To rule out any possible bias in the use of a together with the simultaneous expression of Cas9 and GFP non-targeting control, we also tested a second control plasmid proteins separated by a 2A self-cleaving peptide (Fig. 1b). (LVCas9_dC1/2) targeting two unrelated genomic loci that gen- erate two genomic DSB in two different chromosomes. Results CRISPR-mediated deletion selectively reduces FO products. showed no changes to cell viability in transduced A673 cells We first examined the capacity of LVCas9_EF to generate EF (Supplementary Table 1 and Supplementary Fig. 2g). deletions in A673 and also RD-ES Ewing sarcoma cells, which harbor different EF isoforms. The pLV-U6sgNT-EFSCas9)33 CRISPR FO targeting does not impair genomic stability. We single non-targeting vector (hereafter termed LVCas9_NT) was next evaluated whether our CRISPR-mediated EF targeting used as a control (Supplementary Table 1). PCR analysis of the strategy induces collateral genomic alterations. We used as wild- genomic regions spanning the intronic cleavage sites type control clinically-relevant healthy hMSC, which are NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x considered the cell-of-origin for human sarcomas35. Sequencing FO deletion controls tumor growth in a xenograft model. We analysis of EF-targeted hMSCs confirmed the presence of a chose adenovirus (AdV) vectors to test the in vivo applications of repertoire of indels (~55% and 68%, EWSR1 and FLI1, respec- the targeting approach because of their high gene transfer effi- tively) at the targeted regions (Supplementary Fig. 4a). To assess ciency, their ability to infect cancer cells, their capacity for large possible side-effects that could affect proliferation of healthy cells, DNA transgenes (allowing co-delivery of sgRNAs and Cas9 we treated hMSCs with LVCas9_NT or LVCas9_EF, finding no within single vector particles) and also because they do not difference in proliferation rates in long-term culture (Supple- readily integrate into host genomes38. To test whether AdV-based mentary Fig. 4b). Also, hMSC transduced with LVCas9_EF did in vivo delivery of the FO-targeting CRISPR machinery can not produce tumors when injected into immunosuppressed mice. control tumor growth, we subcutaneously injected A673 cells into G-banding karyotype analysis 7 days after transduction revealed immunodeficient mice and allowed tumors to develop for 10 days no numerical or structural abnormalities in hMSC, irrespective of (~150 mm3 in size) (Fig. 3a). Tumors were then injected with 100 the vector used (Supplementary Fig. 4c), and high-density gen- μL of PBS containing 2.5 × 109 plaque-forming units (pfu) of the ome-wide array comparative genomic hybridization (aCGH) AdCas9_EF vector containing the same cassette as described analysis confirmed no large copy number deletions across the above for the LV vector (sgE3 and sgF8 together with Cas9 and genome (Supplementary Fig. 4d). hMSCs were also negative for GFP proteins), or the equivalent non-targeting AdCas9_NT EWSR1-FLI1 translocation products by FISH and PCR analysis at control vector, or PBS, at day 10, 13, 16 and 19 (Fig. 3a). Results 15- and 30-days p.t. (Supplementary Fig. 4e, f and Table 1). revealed that compared with AdCas9_NT- or PBS-treated mice, Finally, qRT-PCR analysis of edited hMSCs confirmed non- tumor size in AdCas9_EF-treated mice was reduced by ~70% at significant variation in the levels of EWSR1 and FLI1 expression sacrifice (1345.2 ± 685.2 and 1144.0 ± 213.1 vs 375.7 ± 114.9 mm3, (Supplementary Fig. 4g). To test for potential off-target cleavage respectively, p < 0.01) (Fig. 3b, c). Immunohistochemistry analysis activity, we performed next-generation sequencing (NGS) of PCR showed a robust decrease in the number of Ki67+ cells in EF- amplicons spanning the 50 more probable in silico-predicted off- targeted tumor sections (90% and 88% vs 12%, p < 0.001) and an target sites of sgE3 and sgF8. The threshold percentage for off- increase in the number of apoptotic cells (9% and 10% vs 45%, p target activity was set at 0.10%, since this was the highest per- < 0.001) (Fig. 3d, e). Of note, 40% of xenografted mice treated centage of indel-containing reads in control samples, considering with AdCas9_EF survived beyond day 80, whereas all PBS- or as noise indel-containing reads below this percentage36. For every AdCas9_NT-treated mice succumbed to the disease by day 40 off-target site analyzed, indel-containing reads represented less (Fig. 3f). Hematoxylin and eosin (H&E) staining of tumors than 0.10% of the total, with the exception of one predicted site revealed a significantly lower number of viable tumor cells and (Supplementary Table 3, Supplementary Fig. 4h). Although this more extensive necrotic regions in EF-targeted tumors (Supple- site showed indel-containing reads at a slightly higher frequency mentary Fig. 5a). Immunohistochemistry for Cas9 confirmed (∼0.12%) than the threshold, the sequence variations found were adenoviral delivery into the tumors (Fig. 3d). Immunohisto- also present in both controls and edited samples, suggesting that chemostry analysis of the tumors from AdCas9_EF-treated mice they were not bona fide Cas9 off-targets. Thus, targeted NGS showed the colocalization of adenovirus expression (Cas9 ruled out mutations in EF-targeted hMSCs, A673, and RD-ES immunostaining) with high and low expression of caspase-3 and cells harvested 7 days after LVCas9_EF transduction. Overall, Ki67, respectively (Fig. 3g). Because we used the Hds:Athymic these data show that the therapeutic targeting of the EF FO seems Nude-Foxn1nu mouse strain, which lacks T cells and shows a highly specific and does not interfere with non-rearranged alleles partial defect in B cell development (with a weakened innate in human control healthy cells. immune system), we tested for the presence of tumor-infiltrating CD45 + leukocyte cells. Results showed that the necrotic region was surrounded by tumor-infiltrating murine CD45 + leukocytes Targeting of a driver FO inhibits tumor cell growth in vitro. (Fig. 3g). Targeted deep-sequencing analysis of the tumors at We utilized in vitro assays to examine the functional con- sacrifice revealed that EF-targeted xenografted tumors analyzed sequences of targeting EF. Transduction with LVCas9_EF, but 6 weeks after AdV injection were composed of non-edited (85%) not with LVCas9_NT, resulted in a 65.5% and 93.9% decrease or partially-edited (one locus; 12% of EWSR1 or 3% of FLI1) in A673 and RD-ES cell growth, respectively (Fig. 2a, b), and a cancer cells (Supplementary Table 2e, f). No cells harboring an EF 60.5% and 70.3% decrease, respectively, in clonogenic capacity deletion were present in the tumors suggesting that CRISPR- (Fig. 2d, e). No changes were found in LVCas9_EF-transduced mediated abrogation of EF is sufficient to inhibit survival of control U2OS cells (non-EWSR1-FLI1) (Fig. 2c, f). Consistent Ewing sarcoma cells in vivo. Furthermore, targeted deep- with the growth phenotype observations, EF-targeted cells had sequencing of PCR amplicons covering the 50 most probable higher levels (5–8-fold) of apoptosis, measured both by pro- predicted off-target sites associated with sgE3 and sgF8 ruled out pidium iodide staining (subG1 cells, Fig. 2g) and caspase-3 mutations in EF-targeted xenografted tumors analyzed 6 weeks analysis (Fig. 2h), than control transduced cells, suggesting that after AdV injection (Supplementary Table 3). CRISPR-mediated abrogation of EF is sufficient to inhibit the survival, proliferation, and clonogenicity of EF-expressing Ewing sarcoma cells in vitro. As the induction of two DSBs in FO deletion controls tumor growth in PDX models. To further the same chromosome has been related to inversion events37, investigate the utility of the approach, we used three patient- we checked for the presence of this type of rearrangement in derived xenograft (PDX) models of Ewing sarcoma. The models A673 cells. PCR analysis revealed the presence of inversion were established by subcutaneous implantation of Ewing sarcoma products at 3 days p.t.; however, inversion carrier cells were PDXs into immunodeficient mice, which were allowed to develop lost during cell culture and no inversion products were for 12 days (tumors ~150 mm3 in size) (Fig. 4a). FISH analysis detectable at day 21 p.t. (Supplementary Fig. 2h). In silico showed the presence of the t(11;22) translocation (Supplementary analysis predicted that an inversion of the FO targeted region Fig. 5b). We next examined the capacity of FO targeted deletions will produce a truncated protein similar to that induced by to reduce PDX growth by measuring tumor size. As described DNA deletion (Supplementary Fig. 3a), which is compatible earlier, EWSR1-FLI1 targeted therapy was administered by ade- with a similar mechanism of cell death of inversion carrier noviral delivery using 2.5 × 109 pfu of the AdCas9_EF in 100 μL cells. of PBS (or a PBS control) at day 12, 14, 16 and 19 (Fig. 4a). 4 NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x ARTICLE a A673 b RD-ES c U2OS ns 8 2.0 2.0 LVCas9_NT ** LVCas9_NT *** LVCas9_NT Cell number (x1010) Cell number (x1010) Cell number (x109) LVCas9_EF LVCas9_EF LVCas9_EF 6 1.5 1.5 4 1.0 1.0 2 * 0.5 0.5 *** * * ** ** 0 0 * 0 2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 Time (days) Time (days) Time (days) d A673 e RD-ES f U2OS LVCas9_NT LVCas9_EF LVCas9_NT LVCas9_EF LVCas9_NT LVCas9_EF *** ** ns 150 150 150 Number of colonies Number of colonies Number of colonies 100 100 100 50 50 50 0 0 0 LVCas9_NT LVCas9_EF LVCas9_NT LVCas9_EF LVCas9_NT LVCas9_EF g h LVCas9_NT *** LVCas9_NT 2.3% 50 25 * Caspase 3 positives 40 cells per field (%) 20 Apoptosis (%) 30 15 G1 G2 10 20 LVCas9_EF LVCas9_EF 21% 5 10 0 0 LVCas9_NT LVCas9_EF LVCas9_NT LVCas9_EF G1 G2 Fig. 2 CRISPR-mediated targeting of EWSR1-FLI1 inhibits in vitro cell growth. a–c Growth rate assay curves of A673 (**p = 0.01), RD-ES (***p = 0.0005) and U2OS edited (LVCas9_EF) and control (LVCas9_NT) cells. (n = 6 independent experiments). d–f Representative crystal violet staining of A673 (***p = 0.0003), RD-ES (**p = 0.0017) and U2OS experimental and control cells. Bottom panels show the statistical analysis of the number of colonies. (n = 3 independent experiments). g Left panel shows DNA profile analysis by propidium iodide staining and flow cytometry. FlowJo v10.0.7 flow cytometry analysis of DNA fragmentation by subG1 population (cells with fragmented DNA) quantification. The percentage of cellular apoptosis was calculated using the percentage of the subG1 peak. Right panel shows graphical representation of subG1 analysis. (n = 4 independent experiments) *p = 0.027. h Representative images of caspase-3-immunostained A673 cells. Scale bars, 50 μm. Right panel shows the percentage of caspase-3-positive cells per field analyzed. (n = 520 (LVCas9_NT) and n = 613 (LVCas9-EF) cells examined over three independent experiments), ***p = 1.07e-9. The error bars indicate the s.e.m. for the averages across the multiple experiments; p-values are represented (ns non-significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). Statistical are two-tailed unpaired t-test. Results showed that tumors were >74% smaller in AdCas9_EF- confirmed adenoviral delivery into the tumors (Fig. 4d). Of note, treated mice at sacrifice than in PBS-treated mice (334.87 ± 28.01 100% of PDX mice treated with AdCas9_EF survived beyond day vs 1321.70 ± 249.77 mm3, p < 0.001) (Fig. 4b, c). Immunohis- 52% and 50% beyond day 70, whereas all PBS control mice tochemistry analysis of tumor sections showed significantly fewer succumbed to the disease by day 40 (Fig. 4f). Taken together, Ki67+ cells in EF-targeted tumors (18% vs 73%, p < 0.001) and an these results show that in vivo adenoviral delivery of EF-targeting increased number of apoptotic cells (57% vs 1.5%, p < 0.001) CRISPR components controls sarcoma growth. (Fig. 4d, e). H&E staining also revealed a significantly lower number of viable tumor cells and more extensive necrotic regions Combined deletion and chemotherapy enhances tumor in EF-targeted tumors (Fig. 4d). Immunohistochemistry for Cas9 regression. We next investigated the possible synergy between NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x a b c PBS AdCas9_NT 2500 PBS Tumour volume (mm3) AdCas9_EF AdCas9_NT AdCas9_NT 2000 AdCas9_EF Ewing sarcoma 1500 Begining of cell treatment 1000 AdCas9_EF Subcutaneous Begining of End of Analysis 500 injection treatment treatment 0 * * ** ** 0 5 10 15 19 25 0 5 10 15 20 25 Time (days) Time (days) d e ns *** f *** Ki67 Positive cells (%) Ki67 Caspase-3 Cas9 100 PBS 80 100 60 AdCas9_NT Survival (%) 40 AdCas9_EF PBS 20 0 50 S NT EF PB 9_ 9_ * s s Ca Ca Ad Ad Caspase-3 positive cells (%) *** 100 ns *** AdCas9_NT 80 0 60 0 20 40 60 80 100 40 Time (days) 20 0 PB S 9_ NT 9_ EF g Cas9 Ki67 Caspase-3 CD45 C as C as AdCas9_EF Ad Ad ns Cas9 positive cells (%) 100 80 60 40 20 0 S PB NT EF 9_ 9_ C as C as Ad Ad Fig. 3 Deletion of EWSR1-FLI1 inhibits tumor growth in xenografted models. a Diagram showing the approach for the in vivo xenograft treatment. A673 cells were subcutaneously injected into immunodeficient mice and tumors were allowed to develop (~150 mm3 in size). Tumors were then injected with AdCas9_EF edition vector, AdCas9_NT control vector or PBS at days 10, 13, 16, and 19. Mice were sacrificed when tumor volume reached 1500 mm3. b Tumor growth over 23 days of analysis. (PBS n = 6; AdCas9_NT n = 6; AdCas9_EF n = 15 animals), **p = 0.004. c Images of representative tumors. d Representative immunostaining for Ki-67, caspase-3 and Cas9 in A673 experimental and control tumors (n = 3 independent samples). Scale bars, 50 μm. e Statistical analysis of the percentage of Ki-67- (PBS vs AdCas9_EF ***p = 1e-8, AdCas9_NT vs AdCas9_EF ***p = 6e-9), caspase-3- (PBS vs AdCas9_EF ***p = 1e-5, AdCas9_NT vs AdCas9_EF ***p = 5e-5) and Cas9-positive cells. f Kaplan–Meier survival curve comparing mice treated with experimental and control AdV or PBS. (PBS n = 4; AdCas9_NT n = 5; AdCas9_EF n = 10 animals), *p = 0.046. g Representative immunostaining for Cas9, Ki-67, caspase-3, and CD45 in an AdCas9_EF-treated xenografted tumor. The error bars indicate the s.e.m. for the averages across the multiple experiments; p-values are represented (ns non-significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). A two-tailed unpaired t-test was used for statistical analysis of b, d, and log-rank test for f. our genomic editing approach to efficiently eliminate cancer cells showed that mice treated with combined therapy showed a and a standard-of-care chemotherapeutic option for Ewing sar- greater reduction in tumor size at sacrifice (92.12%, 115.28 ± coma with doxorubicin. We examined the additive effects of 28.35 mm3) than mice treated with monotherapy (83.7%, 237.4 ± combined doxorubicin and EF deletion both in vitro and in vivo. 47.39 mm3 AdCas9_EF or 70.4%, 433.05 ± 56.91 mm3 doxor- Corresponding FO deletion and treatment of A673 cells with 0.05 ubicin) as compared with control mice (1463.82 ± 278.17 mm3) μg/ml doxorubicin resulted in a greater reduction of cell viability (Fig. 5c). These results indicate that combined therapy is more (50%) than single treatments (39% or 42% with AdCas9_EF or effective than monotherapy. Of note, 66% of xenografted mice doxorubicin treatment alone, respectively) (Fig. 5a). For in vivo treated with combined therapy survived beyond day 80 compared analysis, we utilized the xenograft models generated as described with 33% of AdCas9_EF-treated mice. By contrast, all the mice earlier, divided into four treatment groups. Mice were treated that received doxorubicin monotherapy succumbed to the disease with 1.5 mg/kg doxorubicin (intraperitoneal injection, weekly, for by day 64 (Fig. 5d). two weeks) or with 2.5 × 109 pfu of the AdCas9_EF or AdCas9_NT virus in PBS (intratumour injection, at day 9, 12, 15, Validation as a method for elimination of FO-addicted cells. and 18) (Fig. 5b). Three groups received monotherapy of To validate the editing strategy as a universal strategy for elim- AdCas9_NT, doxorubicin or AdCas9_EF, while the remaining ination of FO-driven cancer cells, we applied the same approach group was treated with combined therapy (doxorubicin + in a cellular model harboring BCR-ABL1 (BA), a classical tyro- AdCas9_EF), and animals were monitored for 80 days. Results sine kinase FO hallmark of chronic myeloid leukemia (CML). 6 NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x ARTICLE a b c AdCas9_EF 1750 PBS *** Tumor Volume (mm3) PBS 1500 AdCas9_EF 1250 *** Ewing sarcoma 1000 PDX Begining of ****** AdCas9_EF 750 treatment *** Subcutaneous Begining of End of Analysis 500 *** *** implantation treatment treatment ** 250 0 0 5 12 15 19 25 30 35 40 0 10 20 30 40 50 Time (days) Time (days) d e Caspase-3 positive cells (%) *** *** *** Cas9 positive cells (%) 100 100 Ki67 Positive cells(%) 100 75 75 75 H&E Ki67 Caspase-3 Cas9 50 50 50 25 25 25 0 0 0 PBS S S S EF EF EF PB PB PB 9_ 9_ 9_ as as as C C C Ad Ad Ad f AdCas9_EF 100 Survival (%) 75 *** 50 PBS 25 AdCas9_EF 0 0 10 20 30 40 50 60 70 80 Time (days) Fig. 4 Deletion of EWSR1-FLI1 inhibits tumor growth in PDX models. a Diagram showing the approach for the AdV-based in vivo treatment. PDXs were implanted into immunosuppressed mice and tumors were allowed to develop (~150 mm3 in size). Tumors were then injected with AdCas9_EF edition vector or PBS control vector at days 12, 14, 16, and 19. Mice were sacrificed when tumor volume reached 1500 mm3. b Tumor growth over the 40 days following subcutaneous PDX implantation. (PBS n = 3; AdCas9_sgEF n = 9 animals) (***p = 3e-5). c Representative tumors of control and experimental mice sacrificed 40 days after PDX implantation. d Representative images of histological H&E staining, Ki-67, caspase-3, and Cas9 immunostaining of A673 experimental and control cells. Scale bars, 500 μm (4×) or 50 μm (20×). e Percentage of Ki-67-(***p = 3e-6), caspase-3-(***p = 3e-5) and Cas9-positive cells per field analyzed (***p = 4e-6). (n = 3 independent samples). f Kaplan–Meier survival curve comparing mice treated with experimental and control AdV. (PBS n = 3; AdCas9_sgEF n = 9 animals), ***p = 0.0002. Plot shows medians and ranges; error bars indicate the s.e.m. for the averages across the multiple experiments; p-values are represented (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). A two-tailed unpaired t-test was used for statistical analysis of b, e, and log-rank test for f. Four sgRNAs targeting BCR intron 8 and ABL intron 1 regions colony formation and the highest percentage of apoptosis rate were designed to induce a 133.9 kb deletion of the BA FO this combination was chosen for further study. For subsequent (Supplementary Table 1), resulting in an extensive deletion of the targeting of BA, sgB8.1 and sgA1.1 were cloned into an all-in-one BCR transactivation domain and a frameshift alteration of the expression plasmid34 (pLVCas9_BA). Targeted deep-sequencing entire ABL1 DNA-binding domain (Fig. 6a and Supplementary of sgB8.1 or sgA1.1-targeted K562 cells revealed 85% and 82% Fig. 3b). Four sgRNA combinations (sgB8.1-sgA1.1, sgB8.1- indels in BCR and ABL1 on-target sites, respectively (Supple- sgA1.2, sgB8.2sgA1.1, and sgB8.2-sgA1.2) were cloned into the mentary Table 2). Similarly, targeted deep-sequencing of PCR pLV-U6BH1A-C9G vector as before, and were electroporated into amplicons covering the most probable predicted off-target sites the BA-expressing CML cell line K562 (Fig. 6b). Electroporation associated with sgB8.1 and sgA1.1 ruled out mutations in BA- was chosen as the delivery method because of the low trans- targeted K562 cells after pLVCas9_BA electroporation (Supple- duction efficiency of lentiviral vectors in K562 cells39. All four mentary Table 3). qRT-PCR analysis of FO expression at day 2 sgRNA combinations efficiently abrogated the expression of BA post-electroporation (p.e.) of pLVCas9_BA edited K562 cells used as confirmed by RT-PCR and Sanger sequencing (Fig. 6c). For to estimate rates of deletion revealed a significant 5.0-fold any given sgRNA combination, electroporated K562 cells showed decrease in FO expression (Fig. 6e). We also analyzed the pre- a significant decrease (∼85%) in clonogenic capacity in vitro sence of inversions in K562 cells as before. PCR analysis revealed (Fig. 6d) concomitant with an increase in apoptosis (3% vs 15%, the presence of inversion products at 2 days p.e.; however, p < 0.001) relative to equivalent cells electroporated with a non- inversion carrier cells were lost during cell culture and no targeting vector (Supplementary Fig. 6a). The use of a control inversion products were detectable at day 7 p.e. (Supplementary plasmid (LVCas9_dC3/4) targeting two unrelated genomic loci in Fig. 6d). In silico analysis showed that an inversion of the FO two different chromosomes ruled out any effects on cell viability targeted region will produce a truncated protein similar to the one in K562-treated cells due to the induction of two DSBs (Sup- induced by DNA deletion (Supplementary Fig. 3b). We used cord plementary Table 1 and Supplementary Fig. 6b, c). Because blood-derived human hematopoietic progenitor hCD34+ cells as cleavage with sgB8.1-sgA1.1 resulted in the highest reduction in a control cell of origin for CML. In vitro analysis of targeted NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x a b O OH O OH OH H O O OH O O DOX 1.0 OH NH2 Absorbance (A450-A600) 0.9 AdCas9_EF AdCas9_NT 0.8 ** ** ** Ewing sarcoma 0.7 *** cell 0.6 Subcutaneous Begining of End of Analysis injection treatment treatment 0.5 0 5 10 15 19 25 0.4 Time (days) T EF X T EF N N O 9_ 9_ 9_ 9_ D as as as as C C C C LV LV LV LV + + X X O O D D c d 100 2000 Tumour Volume (mm3) 75 Survival (%) 1500 AdCas9_NT AdCas9_NT Treatment 50 AdCas9_EF 1000 AdCas9_EF start DOX DOX 500 DOX + AdCas9_EF 25 DOX + AdCas9_EF ** *** 0 *** 0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 0 10 20 30 40 50 60 70 80 90 Time (days) Time (days) Fig. 5 Combined approach of EWSR1-FLI1 deletion with doxorubicin inhibits tumor growth in xenograft models. a WST-1 cell proliferation analysis of A673 cell treated with CRISPR-deletion or doxorubicin as monotherapy, or as combined therapy and controls (n = 3 independent experiments) (LVCas9_NT vs LVCas9_EF **p = 0.01, LVCas9_NT vs Dox **p = 0.002, LVCas9_NT vs Dox + LVCas9_NT **p = 0.005, LVCas9_NT vs Dox + LVCas9_EF ***p = 0.0001). b Diagram showing the approach for the in vivo xenograft treatment. A673 cells were subcutaneously injected into immunodeficient mice and tumors were allowed to develop (~150 mm3 in size). Tumors were then injected with AdCas9_EF edition vector, doxorubicin (DOX), AdCas9_NT control vector or their combination at days 9, 12, 15, and 18. Mice were sacrificed when tumor volume reached 1500 mm3. c Tumor growth over the 19 days following subcutaneous cells implantation. (AdCas9_NT n = 5; AdCas9_sgEF n = 6; DOX n = 6, DOX + AdCas9_EF n = 6 animals) (AdCas9_NT vs DOX **p = 0.003, AdCas9_NT vs AdCas9_EF ***p = 9e-4, AdCas9_NT vs DOX + AdCas9_EF ***p = 5e-4). d Kaplan–Meier survival curve comparing mice treated with CRISPR-deletion or DOX as monotherapy, or as combined therapy and controls. (AdCas9_NT n = 5; AdCas9_sgEF n = 6; DOX n = 6, DOX + AdCas9_EF n = 6 animals) (DOX + AdCas9_EF vs AdCas9_EF *p = 0.035, DOX + AdCas9_EF vs DOX **p = 0.003, DOX + AdCas9_EF vs AdCas9_NT ***p = 0.0007). Plot shows medians and ranges; error bars indicate the s.e.m. for the averages across the multiple experiments; p-values are represented (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). A two-tailed unpaired t-test was used for statistical analysis of a, c, and Log- rank test for d. CD34 + cells revealed no difference in proliferation in long- to the disease by day 34 (Fig. 6i). As observed in the Ewing term culture, suggesting no production of collateral cancer- sarcoma model, the efficiency of the AdCas9_BA editing vector driven genomic alterations (Supplementary Fig. 6e). No BCR- was also associated with a robust decrease in the number of ABL1 translocation products were detected in cells by PCR or Ki67+ cells (82% vs 17%, p < 0.001) and an increase in apop- FISH assays conducted at 15- and 30-days p.e. (Supplementary totic cells (2.5% vs 10%, p < 0.05) (Supplementary Fig. 6h, i). Fig. 6f, g, Supplementary Table 1). We then evaluated whether H&E staining also revealed a significantly lower number of AdV delivery of the BA-targeting CRISPR machinery (sgB8.1- viable tumor cells and more extensive necrotic regions in BA- sgA1.1) also controls the growth of BA-driven tumors in vivo. targeted tumors (Supplementary Fig. 6h). A combinatorial Accordingly, K562 cells were subcutaneously injected into approach using BA deletion with a standard-of-care therapeutic immunodeficient mice and tumors were allowed to develop for option in CML (imatinib, 0.105 μg/ml) resulted in a significant 15 days (~150 mm3 in size) (Fig. 6f). Tumors were then injected reduction in K562 cell viability compared with any of the with 2.5 × 109 pfu of the AdCas9_BA editing vector or the treatments in monotherapy (Fig. 6j). Targeted deep-sequencing AdCas9_NT control vector at day 15, 18, and 21. Adenoviral analysis of the tumors at sacrifice revealed that BA-targeted delivery of AdCas9_BA profoundly inhibited tumor growth, xenografted tumors analyzed 6 weeks after AdV injection were resulting in an 88% decrease in tumor size at sacrifice (281 ± 63 composed of non-edited (94%) or partially-edited (one locus; vs 1152 ± 158 mm3 (p < 0.001) compared with control tumors 3% of BCR or ABL1) cancer cells (Supplementary Table 2g, h). (Fig. 6g, h). Strikingly, 50% and 22% of xenografted mice Taken together, these results confirm the efficacy of CRISPR- treated with AdCas9_BA were alive after 38 and 64 days, mediated targeting of sarcoma (EF)- and leukemia (BA)-asso- respectively, whereas all AdCas9_NT-treated mice succumbed ciated FO for cancer cell-directed elimination. 8 NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x ARTICLE a b BCR-ABL1 U6 H1 EFS Cas9 eGFP Wpre LTR sgB sgA P2A LTR sgRNAs sgB8.1 sgA1.1 sgB8.2 sgA1.2 Exons 7 8 9 10 11 14 1 2 3 4 5 6 7 8 9 10 11 LVCas9_BA DNA Gene BCR ABL-1 RNA Protein Analysis CML CML cell transduced cell c d e 1 2 1 2 . . . . A1 A1 A1 A1 sg sg sg sg 1- 1- 2- 2- 150 . . . . B8 B8 B8 B8 M sgNT sg sg sg sg bp Number of colonies wt 1000 100 BCR-ABL1 relative 1.5 expression level 700 500 Deletion 1.0 50 *** *** 300 300 *** *** 0.5 ** GAPDH 100 0 0.0 NT BA 2 1 1 2 T 9_ 9_ 1. 1. 1. 1. N as as gA gA gA gA sg C C LV LV -s -s -s -s .1 .1 .2 .2 B8 B8 B8 B8 sg sg sg sg BCR exon 8 ABL-1 exon 2 f g AdCas9_NT AdCas9_BA AdCas9_BA AdCas9_NT CML cell Subcutaneous Begining of End of Analysis injection treatment treatment 0 5 10 15 20 25 30 Time (days) h i j AdCas9_NT 1.0 100 Absorbance (A450-A600) 1500 ns AdCas9_BA *** * Tumor Volume (mm3) AdCas9_NT 0.8 ** 75 Survival (%) AdCas9_BA 1000 Beginning of *** 0.6 treatment *** 50 *** *** 0.4 500 *** *** ** 25 * **** 0.2 0 0 0.0 0 10 20 30 0 10 20 30 40 50 60 70 BA BA 62 T IM T _N N 9_ K5 9_ Time (days) Time (days) 9_ as 9 as as as C C C C LV LV LV LV + + IM IM Discussion development of new therapies are still needed. The ability to FOs are ideal therapeutic targets for the development of new precisely manipulate cancer cell genomes to correct or eliminate directed cancer treatments, owing to their cancer-driving roles, cancer-causing aberrations by highly-efficient CRISPR/Cas9 their restriction to cancer cells and the reliance of tumors on genome editing opens new possibilities to develop FO-targeted them. Unfortunately, FOs are challenging to target directly with options to eliminate cancer cells. In the present study, we describe candidate drugs, and although some successful anti-cancer drugs a simple and efficient genome editing strategy specifically tar- have been developed based on the ability to target FOs40,41, the geting FOs in cancer cells. Our CRISPR/Cas9-based approach NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x Fig. 6 Strategy validation in CML-initiating BCR-ABL model. a Schematic representation of the BCR-ABL1 locus showing the sgRNAs targeting introns 8 of BCR and 1 of ABL1. b Schematic illustration of the vector and the approach for in vitro treatment. c Agarose gel electrophoresis of BCR-ABL RT-PCR products obtained from experimental and control K562 cells electroporated with four combinations of sgRNAs. RT-PCR analysis was done using RNA extracted from cells at day 2 post-nucleofection. Arrows depict the sizes of wild-type (1125 bp) and deleted (458 bp) RT-PCR products. GAPDH was used as an internal control of the RT-PCR reaction. Bottom panel shows a representative chromatogram of Sanger sequencing analysis of the RT-PCR products. d, Representative images and graphical representation of colony formation of K562 experimental and control cells (stained with nitrotetrazolium blue). (n = 3 independent experiments). sgNT vs sgB8.1-agA1.1 ***p = 3e-10, sgNT vs sgB8.1-agA1.2 ***p = 5e-4, sgNT vs sgB8.2-sgA1.1 ***p = 3e-4, sgNT vs sgB8.2-agA1.2 ***p = 1e-4. e BCR1-ABL expression level analysis. Relative expression level of BCR-ABL1 in control (LVCas9_NT) and treated (LVCas9_EF) K562 cells measured by qRT-PCR and normalized to GUSB (n = 6 independent experiments, **p = 0.0020). f Diagram of xenograft production, adenoviral treatment and tumor growth over the 30 days following subcutaneous cell injection and four in vivo adenoviral treatments. (AdCas9_NT n = 10; AdCAas9_sgBA n = 10). g Representative tumors of control and experimental mice. h Tumor growth curve over the 28 days of study. (AdCas9_NT n = 10; AdCas9_sgBA n = 10, ***p = 3e-4). i Kaplan–Meier survival curve comparing mice treated with experimental and control adenoviral vectors (AdCas9_NT n = 10; AdCas9_sgBA n = 10 animals, ****p < 0.0001). j WST-1 cell proliferation analysis of K562 cell treated with CRISPR-deletion or imatinib (IM) as monotherapy, or as combined therapy and controls (n = 3 independent experiments), K562 vs LVCas9_BA ***p = 3.9e-5, K562 vs IM *p = 0.043, K562 vs IM + LVCas9_NT **p = 0.006, K562 vs IM + LVCas9_BA ***p = 4e-6. Plot shows medians and ranges; error bars indicate the s.e.m. for the averages across the multiple experiments; p-values are represented (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). A two-tailed unpaired t-test was used for statistical analysis of d, e, h and j, and Log-rank test for i. induces two targeted intronic DSBs in both genes involved in a (ii) off-target effects, and (iii) in vivo delivery remain technical FO that, importantly, produces a cancer cell-specific genomic limitations. As described in the present study, combinatorial deletion that is dependent on the presence of the FO, and has no approaches using gene editing with other therapeutic options effect on wild-type gene expression in non-cancer cells. By tar- (e.g., chemotherapy) further increases the efficacy of this geting two established FOs, involving either a transcription factor approach, allowing better responses with lower chemotherapy (EF) or a tyrosine kinase (BA), we present a proof-of-concept doses for the elimination of cancer cells. In contrast to che- demonstration supporting the efficacy of genome edition to motherapy agents, which can be damaging to normal tissues, we eliminate cancer cells addicted to FOs. Exploiting an adenoviral show that the CRISPR approach is completely specific for the in vivo delivery of CRISPR components, we further demonstrate elimination of cancer cells43,44. Potential off-target events at that CRISPR/Cas9-mediated deletion in advance PDX cancer unintended genomic sites remain important concerns that must models results in efficient induction of cancer cell death and be investigated rigorously to ensure short- and long-term safety of reduces tumor burden and mortality. Finally, we show that tar- any gene-editing approach. We have used in vitro and in vivo geted CRISPR-based FO deletion combined with chemotherapy assays to study the potential effect of collateral cancer-driver agents has an additive/synergistic effect on cell viability, tumor genomic alterations. Even though our results did not show pro- growth and overall survival when used in xenograft models. In liferation differences, these experiments are limited, and further the long term, this approach may provide an attractive strategy to analysis will be needed to rule out the appearance of cancer- eliminate cancer cells and we believe our findings might have driven off-target modifications. Although the CRISPR-based applicability to other FOs beyond EF and BA. Due to the complex toolbox supports diverse operations ranging from DNA and genomic scenario in cancer cells, including co-existence of other RNA editing to gene expression modulation, delivery remains a oncogenes, tumor suppressor genes, DNA damage response and bottleneck for therapy development45. AdV vectors are a com- DNA repair pathways alterations, a case-by-case FO study could monly utilized delivery vector in mouse models; they are ideal shed light to the applicability of the strategy to eliminate different for delivery of the entire CRISPR/Cas9 system in one vector, types of cancer cells. with high transfection efficacy and they limit potential off-target A recent study has described an approach for targeting FOs effects because their genome does not integrate into the host using CRISPR/Cas9-based editing and the HDR DNA repair genome46–48. Although the initially encountered high immuno- mechanism. The strategy involves directly targeting the patient- genicity inhibited their widespread use to treat genetic disorders, specific breakpoint to introduce a suicide gene into the genome of the overall picture changed when it was recognized that adeno- cancer cells25. Notably, this elegant approach is based on poorly viral infection in a tumor can activate a robust immunogenic efficient and breakpoint-specific design. Our system offers unique response, creating new opportunities in cancer therapy49,50. The advantages over breakpoint-targeted approaches. First, it exploits number of clinical trials using oncolytic AdVs is increasing, and the efficient NHEJ pathway active in all cell types, making the at present more than 32 trials are underway in different phases design simple and highly efficient42, as shown here with high (https://clinicaltrials.gov)51. As in our study, the main delivery efficiency targeting FOs for in vivo elimination of cancer cells pathway used in these clinical trials is local administration with significant decreases in tumor volume and increases in (intratumoral injection), which shows no toxicity at specific doses survival rates. Second, the strategy targets two introns flanking all and, in addition to the therapeutic effect, triggers an antitumor breakpoint regions described in patients, making it a universal immune response with therapeutic effects even on not-injected approach for all cancer cells harboring a given FO irrespective of distal tumors52. Our Hds:Athymic Nude-Foxn1 animal model the FO isoform or the patient-specific breakpoint. For those characterized by partial defects in B cell development shows promiscuous FOs (such as EWSR1) that have been described tumor-infiltrating CD45 + mouse leukocyte cells in the tumor. rearranged with many different fusion partners, a combination of Nevertheless, additional studies on immunocompetent syngeneic the original 5′ sgRNA with a set of pre-designed 3′ sgRNAs will mouse models could yield information about the role of AdVs as cover all the possible FO family31. Third, our approach does not a delivery tool in cancer therapy. In the present study, we used affect exonic sequences or the expression of wild-type alleles adenoviral-based in vivo delivery of the CRISPR/Cas9 compo- involved in the rearrangement. nents as a proof-of-concept; however, we envision that other CRISPR/Cas9-mediated genome editing could be a potential delivery platforms such as AAVs and non-viral (polymeric and platform for cancer treatment; however, (i) targeting efficiencies, liposome-based) vectors could be exploited to deliver the system 10 NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x ARTICLE to tumor cells. Further development of safer and robust delivery Apoptotic SubG1 cell detection. Cell cycle distribution was analyzed by mea- systems and the refinement of genome editing technologies will suring the DNA content using flow cytometry. Exponentially growing transduced cells were pelleted by centrifugation, washed with PBS (Sigma-Aldrich), resus- ultimately ensure optimal targeting efficiencies and safety pended in ice-cold 70% ethanol (Sigma-Aldrich) in PBS for fixation, and were required for the potential clinical application of this cancer cell- maintained at 4 °C for 24 h. Prior to analysis, fixed cells were washed with directed therapy. phosphate-citrate buffer (Sigma-Aldrich) and incubated with propidium iodide Overall, our data demonstrate that NHEJ CRISPR-mediated staining solution (Sigma-Aldrich) containing 100 μg/mL of RNase (Qiagen) for 30 FO deletion is a selective and efficient strategy for cancer cell min. Cellular DNA content was measured using a BD FACSCantoTM platform (BD Biosciences) and analyzed by using FlowJo® software (version 10.0.6, FlowJo). elimination, providing a valuable tool for basic research and could represents a promising strategy with therapeutic potential. PCR, RT-PCR, and qRT-PCR analysis. Genomic DNA and total RNA were extracted using standard procedures with the DNeasy Blood and Tissue Kit or the Methods RNeasy Purification Kit, respectively (Qiagen). DNA and RNA were quantified by sgRNA design and generation of lentiviral constructs. sgRNAs were designed spectrophotometry. cDNA was generated from 1 μg of total RNA using the High- using the online Benchling CRISPR gRNA Design tool (http://www.benchling. Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). PCR and RT- com). The sgRNAs chosen were based on a high specificity rank and a low PCR amplification were performed using Q5 High-Fidelity DNA Polymerase potential off-target score53. The parental Streptococcus pyogenes Cas9 expression (NEB). qRT-PCR was performed in 384-well plates with 2x SYBR Green Master lentiviral single and double sgRNAs expression vectors, pLV-sgRNA-Cas9 and Mix (Applied Biosystems) using a QuantStudio 6 Detection System (Applied pLV-U6#1-H1#2-C9G, respectively, have been described33,34. sgRNAs were cloned Biosystems). Expression levels were normalized to the housekeeping genes using synthetic gBlock fragments (IDT) that were digested with PacI and XhoI ALBUMIN for genomic DNA PCR and GAPDH for RT-PCR or GUSB for qRT- (NEB) to facilitate cloning into the lentiviral backbone. When two different guides PCR. The primers used are listed in Supplementary Table 1. were used, the first was cloned under the control of the U6 promoter and the second was cloned under the control of the H1 promoter. The sequences for Western blotting. Proteins were extracted using standard procedures in the sgRNAs, gBlocks, and primers used are listed in Supplementary Table 1. All presence of protease inhibitors57. Protein lysates were fractionated by SDS-PAGE transformations were performed in Stbl3 bacteria (Thermo Fisher Scientific). and transferred to PVDF membranes (Millipore) using TransFi (ThermoFisher Scientific). Membranes were probed for FLI1 with a mouse monoclonal antibody Cell culture. HEK293T/17 (CRL-11268), A673 (CRL-1598), umbilical cord blood- (1/400; BD Pharmigen). GAPDH was used as a loading control (1/200; Abcam). derived hMSC (PCS-500-010), U2OS (HTB-96), and K562 (CCL-243) cells were Secondary antibodies were HRP-conjugated with a goat anti-mouse IgG (1/1000; purchased from the American Type Culture Collection (ATCC). RD-ES cells were Abcam) and blots were developed with the ECL reagent (GE Healthcare) and a gift from Dr. Javier Alonso (Instituto Salud Carlos III, Madrid). HEK293T/17, exposed to film (Kodak). U2OS, and A673 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Lonza) and K562 and RD-ES cells were maintained in Roswell Park Immunoassays. To detect apoptosis, transduced A673 cells were seeded onto glass Memorial Institute (RPMI) medium (Gibco); all were supplemented with 1% coverslips coated with poly-L-lysine (Cultek). After 72 h, cells were washed twice Glutamax (Life Technologies), 10 mg/ml antibiotics (penicillin and streptomycin, with PBS (Sigma-Aldrich), fixed in 4% paraformaldehyde (PFA; Electron Micro- P/S, Gibco) and 10% fetal bovine serum (FBS, Life Technologies). hMSCs were scope Sci) for 12 min at room temperature (RT), permeabilized with 0.3% Triton cultured in MesenPRO RS media (Thermo Fisher Scientific) supplemented with 1% X-100 (Sigma-Aldrich) in PBS and blocked with 3% normal goat serum (NGS; Glutamax (Life Technologies) and 10 mg/ml antibiotics (P/S, Gibco). hCD34+ Sigma-Aldrich) in PBS for 1 h at RT. Thereafter, samples were incubated overnight were obtained freshly from the umbilical cord blood of healthy donors after at 4 °C with an anti-caspase-3 antibody (1/200; BD) diluted in PBS supplemented informed consent was obtained and after approval by the Cord Blood Bank with 1% NGS, and then with an Alexa Fluor-594-conjugated secondary antibody Transfusion Center of the Community of Madrid. Mononuclear cells were purified (1/500; ThermoFisher Scientific) for 1 h at RT. Finally, samples were counter- by gradient centrifugation (Histopaque, Sigma-Aldrich) and hCD34+ cells were stained with DAPI (Vector Labs), air dried and mounted in Vectashield mounting purified by separation using magnetic beads (Miltenyi Biotec)54. hCD34+ cells medium (Vector Labs). Images were acquired on a Leica DM5500B microscope were cultured in StemSpan SFEM II (StemCell Technologies) containing 100 U/ (Leica Microsystems) with two lasers with excitation at 594 nm (red channel, mL P/S and a cytokine cocktail composed of 100 ng/mL of human stem cell factor caspase-3 detection) and 405 nm (blue channel, nuclear DAPI staining). Data were (hSCF) human FMS-like tyrosine kinase 3 (hFlt3L) and human thrombopoietin collected sequentially at a resolution of 1024 × 1024 pixels and are representative of (hTPO) and 10 ng/mL of human interleukin 3 (hIL3) (all from Preprotech). Cells every experiment analyzed using Cytovision v7.4 software (Leica Biosystems). were cultured at 37 °C at 5% CO2, 5% O2 atmosphere in a humidified incubator. All Tumor tissues were fixed with 4% PFA (Electron Microscopy Sci) in PBS cell lines were periodically tested for mycoplasma contamination. overnight at 4 °C for immunohistochemistry assays58. Tissue sections were deparaffinized, rehydrated, blocked with 3% hydrogen peroxide (Merck) and subjected to heat-induced antigen retrieval. The following antibodies were used at Lentivirus generation, titration, transduction. Recombinant lentiviruses were the indicated dilutions: mouse anti-Ki-67 (DAKO; 1:1.000); rabbit anti-cleaved produced by transient plasmid transfection of HEK293T/17 cells55. Briefly, cells caspase-3 (1:800); rabbit anti-GFP (1:1000), mouse anti-Cas9 (1:1000) and Rabbit were seeded at 1.1 × 107 cells/dish in 15-cm dishes the day before transfection, and anti-CD45 (D3F8Q) (1:200) (all from Cell Signaling Technology). Immunostained were transfected by the calcium-phosphate method using 14.6 and 7.9 μg of tissue images were examined with an Olympus AX70 microscope and processed second-generation packaging plasmids (psPAX2 and pMD.2G, Addgene #12260 with ImageJ to estimate the labeling index (percentage of positively stained and #12259, respectively) and 22.5 μg of the appropriate transfer plasmid nuclear area). depending on the experiment. The medium was collected after 48 h, cleared by low-speed centrifugation, and filtered through 0.45 μm PVDF filters (Millipore). Viral stocks were concentrated by ultracentrifugation in a Beckman LE ultra- Karyotyping. Cell cultures were arrested in metaphase by incubation with Kar- centrifuge using a SW28 rotor at 20,000 × g for 2 h at 4 °C. Pellets containing yoMAX Colcemid (Gibco; 0.1 mg/mL). Cells were treated with a hypotonic solu- lentivirus were air dried and resuspended overnight at 4 °C in 300 μL of sterile tion (0.075 M KCl in ddH2O; Merck) for 30 min at 37 °C and were then fixed with NaCl 0.9% solution (B.Braun). Viral titers were calculated by FACS analysis on Carnoy’s solution (methanol and acetic acid 3:1; Sigma-Aldrich). For GTG transduced HEK293T cells when vectors expressed fluorescent proteins (trans- banding, slides were trypsinized (ThermoFisher Scientific) and stained with duction units/mL) and particles quantified by qPCR in supernatants (particles/mL), Giemsa (ThermoFisher Scientific). Metaphase spreads were analyzed on a Zeiss and were between 107 and 108 TU/mL. Viral aliquots were stored at −80 °C. Target Axioplan microscope with Ikaros v5.20 karyotyping platform (Metasystems cells were re-plated 24 h before transduction and transduced using a multiplicity of GmbH). Between 40 and 100 metaphases were analyzed. infection between 2 and 5. Cells were incubated at 37 °C for 6 h and then viral supernatant were replaced with fresh media. Comparative genomic hybridization arrays. Comparative genome hybridization analysis was performed on a dual color oligonucleotide-based array (SurePrint G3 In vitro colony forming assays. For A673, RD-ES and U2OS cells, colony forming Human CGH Array 8 × 60 K; Agilent) with a 41 kb overall median probe spacing assays were performed 24 h after transduction with the corresponding lentiviral (Agilent). Sample and reference DNA (Agilent) samples were labeled with Cy5 and vectors in six-well plates (500 cells/well)56. Cells were allowed to grow for Cy3 dyes (Agilent), respectively. Hybridization was performed according to the 14–21 days, after which cells were fixed using methanol solution and stained with a manufacturer’s protocols. Arrays were scanned using the G2565BA DNA Micro- 0.5% crystal violet solution (Sigma-Aldrich) and counted. For K562 cells, array Scanner (Agilent Technologies) and data were extracted using Feature methylcellulose (StemCell Technologies) assays were performed by seeding 500 Extraction Software v10.7 (Agilent Technologies). The Aberration Detection cells into 35-mm tissue culture dishes (ThermoFisher Scientific) 24 h after elec- Method 2 (ADM-2) algorithm in Cytogenomics v5.0 (Agilent Technologies) was troporation. Colonies were stained two weeks later with Nitrotetrazolium Blue used to identify contiguous genomic regions corresponding to chromosomal chloride solution (NBT, Sigma-Aldrich) for 1 h at 37 °C and counted. All assays aberrations or copy number variations, which were scored based on the average were performed in triplicate and repeated three times. quality weighted log ratio of the sample and reference channels. NATURE COMMUNICATIONS | (2020)11:5060 | https://doi.org/10.1038/s41467-020-18875-x | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18875-x Fluorescence in situ hybridization. The EWSR1 break-apart (Kreatech), EWSR1- WST-1 was added and plates were incubated for a further 3 h at 37 °C in the FLI1 dual-fusion (Cytocell) and BCR-ABL1 dual-fusion FISH probe (Kreatech) incubator. The absorbance was monitored at 450 nm. FISH probes were used to detect t(11;22) and t(9;22) chromosomal translocations. Tissue sections (5 mm) were deparaffinized in xylene and rehydrated in ethanol Statistical analysis. Statistical analysis was performed using GraphPad Prism (Sigma-Aldrich). Tissue sections were pre-treated in 2-[N-morpholino] ethane- software package (version 7.0, GraphPad Software). Data from three or more sulphonic acid (DAKO), followed by pepsin digestion (DAKO). After dehydration, independent experiments were analyzed by two-tailed Student’s unpaired t-test. the samples were denatured in the presence of the appropriate probe at 66 °C for Statistical details of the experiments can be found in the corresponding figure 10 min and left overnight for hybridization at 37 °C in a hybridizer machine legends. To flag levels of significance, asterisks were used as follow (*) p-value less (DAKO). The slides were then washed with 20xSSC/Tween20 buffer at 63 °C and or equal than 0.05, (**) p-value ≤ 0.01, and (***) p-value ≤ 0.001 and (ns) was used mounted on fluorescence mounting medium (with DAPI). FISH signals were when non-significant differences were observed. manually scored by counting the number of nuclei with split signals across the tissue. FISH images were captured using a CCD camera (Photometrics SenSys camera) connected to a PC running the Zytovision image analysis system (Applied Reporting summary. Further information on research design is available in the Nature Imaging Ltd., UK). Research Reporting Summary linked to this article. Targeted deep sequencing. Potential off-target sites were predicted using the Data availability CRISPR Design web server (http://crispr.mit.edu)53, and Cas-OFFinder (http:// The NGS data have been deposited in the BioProject database under the accession code www.rgenome.net/cas-offinder/)59. Customized PCR primers for the on-target and PRJNA659633. All other data supporting the findings of this study are available within top-50 potential off-target sites to amplify the sgRNA target region were ordered the article and its Supplementary Information files and from the corresponding author from IDT with rhAmpSeq index sequences (Supplementary Table 1). Each locus upon reasonable request. was individually amplified using genomic DNA extracted from the relevant cells. First PCR was performed rhAmpSeq Library Kit (IDT) under the following con- ditions: 95 °C for 10 min, 10 cycles of 95 °C for 15 s and 61 °C for 4 min and 99.5 °C Received: 23 December 2019; Accepted: 16 September 2020; for 15 min. PCR products of all potential off-target loci were subsequently equally pooled and purified with Agencourt AMPure XP beads (Beckman). The PCR products were then subjected to a second PCR with Illumina indexed adaptor primers under the following PCR conditions: 95 °C for 3 min, 18 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s, and 72 °C for 1 min. Second PCR products were purified with Agencourt AMPure XP beads (Beckman) at a PCR product:bead ratio of 1:1. The purified libraries were quantified and sequenced using a 300-bp References paired-end cycle on the Illumina MiSeq sequencing platform. Results were ana- 1. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of lyzed with CRISPR RGEN Tools Cas-Analyser software60. 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Levetzow, von, C. et al. Modeling initiation of Ewing sarcoma in human S.R-P.); AEI/MICIU EXPLORA Project BIO2017-91272-EXP and CaixaImpulse (CI18- neural crest cells. PLoS ONE 6, e19305 (2011). 00017;FuGe) to S.R-P. RT-R. is supported by a postdoctoral fellowship from the Aso- 36. Bonafont, J. et al. Clinically relevant correction of recessive dystrophic ciación Española Contra el Cáncer (AECC). P.M. is supported by the European Research epidermolysis bullosa by dual sgRNA CRISPR/Cas9-mediated gene editing. Council (CoG-2014-646903 and PoC-2018-811220), the Spanish Ministry of Science, Mol. Ther. 27, 986–998 (2019). Innovation and Universities (SAF2016), and the Catalunya Government (SGR330 and 37. Li, J. et al. Efficient inversions and duplications of mammalian regulatory PERIS 2017). J.C.S. is supported by the Spanish Ministry of Science, Innovation and DNA elements and gene clusters by CRISPR/Cas9. J. Mol. Cell Biol. 7, Universities (SAF2017-84248-P) and the Spanish Cell Therapy cooperative research 284–298 (2015). network (TERCEL)(RD16/0011/0011). C.B. is supported by the AECC, Beca FERO, and 38. Fukuhara, H., Ino, Y. & Todo, T. Oncolytic virus therapy: a new era of cancer the ISCIII/FEDER (PI17/01028). P.M. also acknowledges the financial support from the treatment at dawn. Cancer Sci. 107, 1373–1379 (2016). Obra Social La Caixa-Fundaciò Josep Carreras. P.M. is an investigator of the Spanish Cell 39. Abbasalipour, M. et al. Improvement of K562 cell line transduction by FBS Therapy cooperative research network (TERCEL). A.M.C. acknowledges funding from mediated attachment to the cell culture plate. Biomed. Res. Int. 2019, ISCIII-FEDER (CP13/00189) and Xarxa de Bancs de Tumors de Catalunya (XBTC; 9540702–9540706 (2019). sponsored by Pla Director d’Oncologia de Catalunya). We thank Dr Kenneth McCreath 40. Dai, X., Theobard, R., Cheng, H., Xing, M. & Zhang, J. Fusion genes: a for critical reading of the paper. promising tool combating against cancer. Biochim. Biophys. Acta Rev. Cancer 1869, 149–160 (2018). Author contributions 41. Schram, A. M., Chang, M. T., Jonsson, P. & Drilon, A. Fusions in solid R.T-R. and S.R-P. conceived the project, designed and performed experiments, analyzed tumours: diagnostic strategies, targeted therapy, and acquired resistance. Nat. the data and wrote the manuscript. M.M-L., did the experiments, analyzed results, and Rev. Clin. Oncol. 14, 735–748 (2017). wrote the paper. P.M-G., P.P-S.; O.Q-B.; S.G-S.; P.P.; M.C.M. and F.J.M. assisted in the 42. Paquet, D. et al. Efficient introduction of specific homozygous and experiments and collected data. C.B.; H.P.; J.C.S. and P.M. analyzed the data and com- heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 mented on the paper. All authors read and approved the final manuscript. A.M.C. and (2016). J.M. provide the PDX tumors and commented the paper. 43. Di Maio, M. et al. Symptomatic toxicities experienced during anticancer treatment: agreement between patient and physician reporting in three randomized trials. J. Clin. Oncol. 33, 910–915 (2015). Competing interests 44. Kaelin, W. G. Common pitfalls in preclinical cancer target validation. Nat. A patent has been filed relating to the data presented in this research study by S.R-P., Rev. Cancer 17, 425–440 (2017). R.T-R. and M.M-L. (EP18382746.8). The remaining authors declare no competing 45. Lino, C. A., Harper, J. C., Carney, J. P. & Timlin, J. A. Delivering interests. CRISPR: a review of the challenges and approaches. Drug Deliv. 25, 1234–1257 (2018). 46. Horwood, N. J. et al. High-efficiency gene transfer into nontransformed cells: Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41467- utility for studying gene regulation and analysis of potential therapeutic 020-18875-x. targets. Arthritis Res. 4, S215–S225 (2002). 47. Holkers, M. et al. Adenoviral vector DNA for accurate genome editing with Correspondence and requests for materials should be addressed to R.T.-R. or S.R.-P. engineered nucleases. Nat. Methods 11, 1051–1057 (2014). 48. Xu, C. L., Ruan, M. Z. C., Mahajan, V. B. & Tsang, S. H. Viral Delivery Peer review information Nature Communications thanks the anonymous reviewer(s) for Systems for CRISPR. Viruses 11, 28 (2019). their contribution to the peer review of this work. Peer reviewer reports are available. 49. Lopes, A. et al. Oncolytic adenovirus drives specific immune response generated by a poly-epitope pDNA vaccine encoding melanoma Reprints and permission information is available at http://www.nature.com/reprints neoantigens into the tumor site. J. Immunother. Cancer 7, 174 (2019). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in 50. Shaw, A. R. & Suzuki, M. 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