In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system (original) (raw)

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Acknowledgements

We would like to thank M. Fazio, M. Ladanyi, G. Riely, S. Armstrong, and the members of the Ventura, Lowe and Jacks laboratories for discussion and comments. We also thank J. Hollenstein for editing the manuscript, T. Jacks for providing tumour samples from K-RasG12D mice, and the Cytogenetic Core Facility of MSKCC for tissue processing and histology. This work was supported by grants from the Geoffrey Beene Cancer Research Foundation (A.V.), NCI (Cancer Center Support Grant P30 CA008748, E.d.S.), HHMI (S.W.L.), NCI Project Grant (S.W.L.); and by fellowships from the American Italian Cancer Foundation (D.M.), the Foundation Blanceflor Boncompagni Ludovisi, née Bildt (D.M.), and the Jane Coffin Childs Foundation (E.M.). C.P.C. was supported by an NCI training grant.

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Authors and Affiliations

  1. Memorial Sloan Kettering Cancer Center, Cancer Biology and Genetics Program, 1275 York Avenue, New York, New York 10065, USA,
    Danilo Maddalo, Eusebio Manchado, Carla P. Concepcion, Ciro Bonetti, Joana A. Vidigal, Yoon-Chi Han, Paul Ogrodowski, Scott W. Lowe & Andrea Ventura
  2. Weill Cornell Graduate School of Medical Sciences of Cornell University, 1300 York Avenue, New York, 10065, New York, USA
    Carla P. Concepcion
  3. Department of Medical Oncology, Milano-Bicocca University, San Gerardo Hospital, 20052, Via G B Pergolesi 33, Monza, Italy,
    Alessandra Crippa
  4. Memorial Sloan Kettering Cancer Center, Thoracic Pathology and Cytopathology, 1275 York Avenue, New York, New York 10065, USA,
    Natasha Rekhtman
  5. Memorial Sloan Kettering Cancer Center, Molecular Pharmacology Program, 1275 York Avenue, New York, New York 10065, USA,
    Elisa de Stanchina
  6. Howard Hughes Medical Institute, 1275 York Avenue, New York, 10065, New York, USA
    Scott W. Lowe

Authors

  1. Danilo Maddalo
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  2. Eusebio Manchado
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  3. Carla P. Concepcion
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  4. Ciro Bonetti
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  5. Joana A. Vidigal
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  6. Yoon-Chi Han
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  7. Paul Ogrodowski
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  8. Alessandra Crippa
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  9. Natasha Rekhtman
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  10. Elisa de Stanchina
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  11. Scott W. Lowe
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  12. Andrea Ventura
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Contributions

D.M. and A.V. conceived the project, designed and analysed the experiments, and wrote the manuscript. S.W.L. contributed to the interpretation of the results and the writing of the manuscript. D.M. generated and tested the constructs, performed the cell-based experiments, and characterized the Eml4_–_Alk tumours. E.M., D.M., C.B., Y.-C.H. and P.O. performed the in vivo experiments. E.d.S. supervised the crizotinib treatment experiments and analysed the results. J.A.V., D.M., C.P.C. and A.V. microdissected and analysed lung tumours to detect the Eml4_–_Alk inversion. C.B., D.M. and A.C. performed the immunostainings. N.R. reviewed the histopathology.

Corresponding author

Correspondence toAndrea Ventura.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Human and murine Eml4–Alk.

a, Alignment of human EML4 exon 13 and mouse Eml4 exon 14. b, Alignment of the junction between the human EML4–ALK (variant 1) and the predicted murine Eml4–Alk proteins.

Extended Data Figure 2 Induction of the Npm1_–_Alk translocation in NIH/3T3 cells.

a, Schematic of the Npm1–Alk translocation. Red arrows indicate the sites recognized by the sgRNAs. b, Sequences recognized by the sgRNAs and location of primers used to detect the Npm1–Alk and Alk–Npm1 rearrangement (top panel). PCR on genomic DNA extracted from NIH/3T3 co-transfected with pX330 constructs expressing the indicated sgRNAs (middle panel). Sequences of four independent subclones obtained from the PCR products and representative chromatogram (bottom panel). c, Detection of the Npm1_–_Alk fusion transcript by RT–PCR on total RNAs extracted from NIH/3T3 cells co-transfected with the indicated pX330 constructs (left panel). The PCR band was extracted and sequenced to confirm the presence of the correct Npm1_–_Alk junction (bottom-right panel). Representative results from two independent experiments.

Extended Data Figure 3 Comparison of dual and single sgRNA-expressing plasmids.

a, Schematic of pX330 (A) and its derivatives (B_–_E) used in these experiments. NIH/3T3 were transfected with these constructs and lysed to extract total RNA and genomic DNA. b, RNAs were analysed by northern blotting with probes against the Alk (left) or Eml4 (right) sgRNAs. c, d, The DNA samples were subjected to surveyor assays (c), or amplified by PCR to detect the Eml4_–_Alk inversion (d).

Extended Data Figure 4 Induction of the Eml4_–_Alk inversion in primary MEFs using an adenoviral vector expressing Flag–Cas and tandem sgRNAs.

a, Schematic of the adenoviral vectors. b, Immunoblot using an anti-Flag antibody on lysates from MEFs infected with the indicated adenoviruses. c, Small-RNA northerns using probes against sgEml4 and sgAlk on total RNAs from cells infected with Ad-Cas9 or Ad-EA. d, PCR-mediated detection of the Eml4–Alk inversion in MEFs infected with Ad-Cas9 or Ad-EA for the indicated number of days. e, Standard curve generated performing quantitative PCR analysis on genomic DNA containing a known fraction of Eml4–Alk alleles. Average of two independent experiments. f, Quantification of the fraction of MEFs harbouring the Eml4_–_Alk inversion at the indicated time points after infection with Ad-EA or Ad-Cas9. Values are mean of three independent infections ± s.d.

Extended Data Figure 5 Radiologic response of Ad-EA-induced tumours to crizotinib treatment.

µCT images from crizotinib- or vehicle-treated mice at day 0 and after 2 weeks of treatment.

Extended Data Table 1 Mouse cohorts

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Extended Data Table 2 Response to crizotinib treatment

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Extended Data Table 3 Oligonucleotides used in this study

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Extended Data Table 4 Primer pairs and PCR reactions

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Maddalo, D., Manchado, E., Concepcion, C. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.Nature 516, 423–427 (2014). https://doi.org/10.1038/nature13902

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