Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Rapid modelling of cooperating genetic events in cancer through somatic genome editing

Abstract

Cancer is a multistep process that involves mutations and other alterations in oncogenes and tumour suppressor genes1. Genome sequencing studies have identified a large collection of genetic alterations that occur in human cancers2,3,4. However, the determination of which mutations are causally related to tumorigenesis remains a major challenge. Here we describe a novel CRISPR/Cas9-based approach for rapid functional investigation of candidate genes in well-established autochthonous mouse models of cancer. Using a KrasG12D-driven lung cancer model5, we performed functional characterization of a panel of tumour suppressor genes with known loss-of-function alterations in human lung cancer. Cre-dependent somatic activation of oncogenic KrasG12D combined with CRISPR/Cas9-mediated genome editing of tumour suppressor genes resulted in lung adenocarcinomas with distinct histopathological and molecular features. This rapid somatic genome engineering approach enables functional characterization of putative cancer genes in the lung and other tissues using autochthonous mouse models. We anticipate that this approach can be used to systematically dissect the complex catalogue of mutations identified in cancer genome sequencing studies.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Purchase on Springer Link

Instant access to full article PDF

Prices may be subject to local taxes which are calculated during checkout

Figure 1: CRISPR/Cas9-mediated somatic gene editing in an autochthonous mouse model of lung cancer.
Figure 2: Histopathological characterization of tumours from pSECC infected animals.
Figure 3: CRISPR/Cas9 efficiently generates insertions and deletions (indels) in autochthonous tumours.

Similar content being viewed by others

Accession codes

Primary accessions

BioProject

References

  1. Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57–70 (2000)

    Article  CAS  PubMed  Google Scholar 

  2. Imielinski, M. et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 150, 1107–1120 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Govindan, R. et al. Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 150, 1121–1134 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. The Cancer Genome Atlas Research Network Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543–550 (2014)

  5. Jackson, E. L. et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 15, 3243–3248 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. McFadden, D. G. et al. Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing. Cell 156, 1298–1311 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Frese, K. K. & Tuveson, D. A. Maximizing mouse cancer models. Nature Rev. Cancer 7, 645–658 (2007)

    Article  CAS  Google Scholar 

  8. Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Farago, A. F., Snyder, E. L. & Jacks, T. SnapShot: Lung cancer models. Cell 149, 246–246.e1 (2012)

    Article  CAS  PubMed  Google Scholar 

  10. Winslow, M. M. et al. Suppression of lung adenocarcinoma progression by Nkx2-1. Nature 473, 101–104 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. DuPage, M. et al. Endogenous T cell responses to antigens expressed in lung adenocarcinomas delay malignant tumor progression. Cancer Cell 19, 72–85 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature Neurosci. 13, 133–140 (2010)

    Article  CAS  PubMed  Google Scholar 

  13. Rock, J. R. & Hogan, B. L. Epithelial progenitor cells in lung development, maintenance, repair, and disease. Annu. Rev. Cell Dev. Biol. 27, 493–512 (2011)

    Article  CAS  PubMed  Google Scholar 

  14. Song, M. S., Salmena, L. & Pandolfi, P. P. The functions and regulation of the PTEN tumour suppressor. Nature Rev. Mol. Cell Biol. 13, 283–296 (2012)

    Article  CAS  Google Scholar 

  15. Curry, N. L. et al. Pten-null tumors cohabiting the same lung display differential AKT activation and sensitivity to dietary restriction. Cancer Discov 3, 908–921 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Snyder, E. L. et al. Nkx2-1 represses a latent gastric differentiation program in lung adenocarcinoma. Mol. Cell 50, 185–199 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013)

    Article  CAS  PubMed  Google Scholar 

  18. Cheung, A. F. et al. Complete deletion of Apc results in severe polyposis in mice. Oncogene 29, 1857–1864 (2010)

    Article  CAS  PubMed  Google Scholar 

  19. Moon, R. T., Kohn, A. D., De Ferrari, G. V. & Kaykas, A. WNT and β-catenin signalling: diseases and therapies. Nature Rev. Genet. 5, 691–701 (2004)

    Article  CAS  PubMed  Google Scholar 

  20. Pacheco-Pinedo, E. C. et al. Wnt/β-catenin signaling accelerates mouse lung tumorigenesis by imposing an embryonic distal progenitor phenotype on lung epithelium. J. Clin. Invest. 121, 1935–1945 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kormish, J. D., Sinner, D. & Zorn, A. M. Interactions between SOX factors and Wnt/β-catenin signaling in development and disease. Dev. Dyn. 239, 56–68 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Hogan, B. L. et al. Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15, 123–138 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Juan, J., Muraguchi, T., Iezza, G., Sears, R. C. & McMahon, M. Diminished WNT → β-catenin → c-MYC signaling is a barrier for malignant progression of BRAFV600E-induced lung tumors. Genes Dev. 28, 561–575 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnol. 31, 827–832 (2013)

    Article  CAS  Google Scholar 

  25. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnol. 31, 822–826 (2013)

    Article  CAS  Google Scholar 

  26. Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nature Biotechnol. 32, 670–676 (2014)

    Article  CAS  Google Scholar 

  27. Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nature Biotechnol. 32, 677–683 (2014)

    Article  CAS  Google Scholar 

  28. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014)

    Article  ADS  CAS  PubMed  Google Scholar 

  29. Heigwer, F., Kerr, G. & Boutros, M. E-CRISP: fast CRISPR target site identification. Nature Methods 11, 122–123 (2014)

    Article  CAS  PubMed  Google Scholar 

  30. Psarras, S. et al. Gene transfer and genetic modification of embryonic stem cells by Cre- and Cre-PR-expressing MESV-based retroviral vectors. J. Gene Med. 6, 32–42 (2004)

    Article  CAS  PubMed  Google Scholar 

  31. Jackson, E. L. et al. The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer Res. 65, 10280–10288 (2005)

    Article  CAS  PubMed  Google Scholar 

  32. DuPage, M., Dooley, A. L. & Jacks, T. Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nature Protocols 4, 1064–1072 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  34. McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)

    Article  PubMed  PubMed Central  Google Scholar 

  36. Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  37. Breese, M. R. & Liu, Y. NGSUtils: a software suite for analyzing and manipulating next-generation sequencing datasets. Bioinformatics 29, 494–496 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Thorvaldsdóttir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013)

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. McFadden and Y. Soto-Feliciano for critical reading of the manuscript, H. Yin, S. Levine and T. Mason for MiSeq sequencing support, R. Stott, J. Bartlebaugh and C. Shivalila for technical assistance and K. Cormier and C. Condon from the Hope Babette Tang (1983) Histology Facility for technical support. This work was supported by the Howard Hughes Medical Institute, the Ludwig Center for Molecular Oncology at MIT and in part by Cancer Center Support (core) grant P30-CA14051 from the National Cancer Institute. T.P. is supported by the Hope Funds for Cancer Research. T.J. is a Howard Hughes Medical Institute Investigator, the David H. Koch Professor of Biology, and a Daniel K. Ludwig Scholar.

Author information

Authors and Affiliations

Authors

Contributions

F.J.S.-R, T.P. and T.J. designed the study; F.J.S.-R, T.P., R.R., M.R.B. and L.S. performed experiments; T.T. generated KrasLSL-G12D/+; Apcfl/fl data; A.B. conducted bioinformatic analyses; N.S.J. generated GG cells; R.T.B. provided pathology assistance; W.X. gave conceptual advice; F.J.S.-R, T.P. and T.J. wrote the manuscript with comments from all authors.

Corresponding author

Correspondence to Tyler Jacks.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Illumina MiSeq sequence datasets have been deposited into the NCBI repository under BioProjectID PRJNA256245.

Extended data figures and tables

Extended Data Figure 1 In vitro validation of pSECC.

a, The Green-Go Cre-reporter cell line used to validate pSECC lentiviruses in vitro. Upon infection with a Cre-containing lentivirus, such as pSECC, cells become GFP+, allowing for purification of pSECC-containing cells by FACS. Red and blue triangles denote pairs of loxP sites, with red loxP sites being able to recombine only with other red loxP sites and blue loxP sites being able to recombine only with other blue loxP sites. b, Validation of sgPten-pSECC. Numbers below the bands denote quantitation of protein level relative to empty vector control. c, Validation of sgNkx2-1-pSECC in a cell line that expresses Nkx2-1. d, e, Validation of sgTom-pSECC by fluorescence activated cell sorting (FACS). Briefly, a cell line obtained from a KrasLSL-G12D/+; p53fl/fl;Rosa26LSL-tdTomato/LSL-tdTomato mouse was infected with either empty-pSECC (d) or sgTom-pSECC (e) and cultured for 10 days post-infection, after which time the cells were collected and analysed by FACS.

Extended Data Figure 2 In vivo validation of pSECC.

a, Representative H&E and tdTomato IHC staining of serial sections from lung tumours of KrasLSL-G12D/+; p53fl/fl;Rosa26LSL-tdTomato/LSL-tdTomato mice infected with Empty-pSECC. bd, Representative H&E and IHC staining of serial sections from negative (b), mixed (c) and positive (d) lung tumours of KrasLSL-G12D/+; p53fl/fl;Rosa26LSL-tdTomato/LSL-tdTomato mice infected with sgTom-pSECC (n = 6). e, Distribution of lung tumours from all mice infected with sgTom-pSECC (n = 6) that were scored as negative, mixed or positive based on tdTomato IHC.

Extended Data Figure 3 Histological analysis of lung tumours obtained from mice infected with pSECC lentiviruses.

ae, Representative H&E images of lung tumours obtained from mice infected with Empty-pSECC (a), sgTom-pSECC (b), sgNkx2-1-pSECC (c), sgPten-pSECC (d), and sgApc-pSECC (e). f, g, Quantification of tumour burden (total tumour area/total lung area) in KrasLSL-G12D/+ (f) or KrasLSL-G12D/+; p53fl/fl (g) animals 10 weeks after infection with pSECC lentiviruses expressing: control (empty or sgTom, KrasLSL-G12D/+ (n = 4) and KrasLSL-G12D/+; p53fl/fl (n = 7)), sgNkx2-1 (KrasLSL-G12D/+ (n = 2) and KrasLSL-G12D/+; p53fl/fl (n = 6)), sgApc (KrasLSL-G12D/+ (n = 3) and KrasLSL-G12D/+; p53fl/fl (n = 6)) and sgPten (KrasLSL-G12D/+ (n = 4) and KrasLSL-G12D/+; p53fl/fl (n = 3)). h, Quantification of BrdU incorporation (BrdU+ cells per mm2) to assess proliferation of tumour cells from lung tumours in KrasLSL-G12D/+; p53fl/fl animals 10 weeks after infection with pSECC lentiviruses expressing: control (empty or sgTom, n = 4 tumours), sgNkx2-1 (n = 11 tumours), sgApc (n = 10 tumours) and sgPten (n = 15 tumours). Mice were given a pulse of BrdU for 4 h before being euthanized. n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001 obtained from two-sided Student’s t-test. All error bars denote s.e.m.

Extended Data Figure 4 IHC-based analysis of mice infected with sgNkx2-1-pSECC.

ac, Negative (a), mixed (b) and positive (c) lung tumours of mice infected with sgNkx2-1-pSECC. d, Distribution of Nkx2-1 IHC staining status in all sgNkx2-1-pSECC infected animals (n = 8) represented as percent of negative, mixed and positive tumours. Positive tumour, 100% of the tumour cells stained positive for Nkx2-1. Mixed tumour, at least 30% of tumour cells stained positive for Nkx2-1. Negative tumour, < 25% of the tumour cells stained positive for Nkx2-1.

Extended Data Figure 5 IHC-based analysis of mice infected with sgPten-pSECC.

ac, Negative (a), mixed (b) and positive (c) lung tumours of mice infected with sgPten-pSECC (n = 9). Positive tumour, 100% of the tumour cells stained positive for Pten. Mixed tumour, at least 30% of tumour cells stained positive for Pten. Negative tumour, < 25% of the tumour cells stained positive for Pten. Dashed line in b demarcates the positive/negative tumour area.

Extended Data Figure 6 IHC-based analysis of KrasLSL-G12D/+- and KrasLSL-G12D/+; p53fl/fl-sgApc tumours.

a, Representative H&E and IHC staining of serial sections from KrasLSL-G12D/+; p53fl/fl-sgTom (control, denoted as KP-sgTom here), KrasLSL-G12D/+-sgApc (denoted as K-sgApc here) and KrasLSL-G12D/+; p53fl/fl-sgApc (denoted as KP-sgApc here) lung tumours. CCSP, Clara cell secretory protein; SP-C, surfactant protein C. b, Contingency table demonstrating a statistically significantly higher number of β-catenin/Sox9 double-positive tumours in KrasLSL-G12D/+; p53fl/fl-sgApc mice (29/33 tumours, 88%) vs K-sgApc mice (41/58 tumours, 71%) (one-sided chi-square test, P < 0.05). c, Percentage of all tumours that stained positive for nuclear β-catenin that stained positive or negative for Sox9 in KrasLSL-G12D/+- and KrasLSL-G12D/+; p53fl/fl-sgApc mice. d, Contingency table demonstrating a statistically significantly higher number of tumours with Nkx2-1 low/negative areas (which are also SP-C low/negative) in sgApc-pSECC animals compared to sgTom-pSECC control animals (two-sided Fisher’s exact test, P < 0.0001). e, Representative IHC staining of serial sections from an Nkx2-1 Low/Neg lung tumour obtained from a KrasLSL-G12D/+; Apcfl/fl mouse 18 weeks after infection with Adeno-Cre. Inset shows Sox9 staining. Low/neg = tumour that had areas with clear downregulation or complete loss of Nkx2-1 or SP-C as assessed by IHC staining.

Extended Data Figure 7 Representative examples of indels observed in lungs and tumours from mice infected with pSECC lentiviruses.

ac, Representative indels observed in the Nkx2-1 (a), Pten (b) and Apc (c) locus from sgNkx2-1T1, sgPtenL1 and sgApcT3 samples, respectively. Left panel, details of sequence alignments around the PAM sequence. Right panel, overview of sequence alignments around the PAM sequence. Deletions and insertions are highlighted in black and purple bars, respectively. Inset in a depicts a magnification of an insertion. d, Distribution of indels (in-frame insertions, frameshift insertions, in-frame deletions and frameshift deletions) observed in samples from mice infected with sgNkx2-1-pSECC, sgPten-pSECC and sgApc-pSECC. Amp, mutations across whole PCR amplicon; PAM, mutations across 7 base pair region upstream of the PAM sequence. e, Table summarizing percentages of indels from total mutant reads (left percentage indicates Amp (mutations across whole PCR amplicon) and right percentage indicates PAM (mutations across 7 base pair region upstream of the PAM sequence). All error bars denote s.e.m.

Extended Data Figure 8 Off-target analysis.

ai, Analysis of off-target editing for sgNkx2-1 (ac), sgPten (df) and sgApc (gi). Briefly, potential off-target cutting at the top three predicted off-target sites (obtained from (http://crispr.mit.edu/); see Supplementary Table 2) for each sgRNA was assayed by Illumina MiSeq. Each plot corresponds to the fraction of bases mutated per position in 10 bp flanks on either side of the PAM sequence (highlighted in red). Samples were obtained from entire lobes (L) from mice 10 weeks after infection with pSECC lentiviruses expressing sgNkx2-1, sgPten, sgApc or sgTom (control).

Supplementary information

Supplementary Information.

This file contains the reference sequences for Nkx2.1, Pten, Apc and top three predicted off-targets of sgNkx2.1, sgPten and sgApc. (PDF 132 kb)

Supplementary Table 1

This file contains the sgRNA sequences and Primer sequences (XLSX 44 kb)

Supplementary Table 2

The file contains the top 25 predicted off-targets for sgNkx2.1, sgPten and sgApc. Score is likelihood of off-target binding. (XLSX 52 kb)

Supplementary Table 3

This file contains the MiSeq counts and quantification of indels (in-frame insertions, frameshift insertions, in-frame deletions and frameshift deletions) observed in purity-corrected samples from mice infected with sgApc-pSECC, sgNkx2.1-pSECC and sgPten-pSECC. (XLSX 29 kb)

Supplementary Table 4

This file contains the read mapping statistics. (XLSX 13 kb)

Supplementary Table 5

This file contains the mutation calls for Nkx2.1 and related OTs. (XLSX 843 kb)

Supplementary Table 6

This file contains the mutation calls for Pten and related OTs. (XLSX 716 kb)

Supplementary Table 7

The file contains the mutation calls for Apc and related OTs. (XLSX 781 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sánchez-Rivera, F., Papagiannakopoulos, T., Romero, R. et al. Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature 516, 428–431 (2014). https://doi.org/10.1038/nature13906

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13906

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer