The prokaryotic type II CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR-associated 9) system is rapidly revolutionizing the field of genetic engineering, allowing researchers to alter the genomes of a large range of organisms with relative ease. Experimental approaches based on this versatile technology have the potential to transform the field of cancer genetics. Here, we review current approaches for functional studies of cancer genes that are based on CRISPR–Cas, with emphasis on their applicability for the development of next-generation models of human cancer.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57–70 (2000).
Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M. A. & Kucherlapati, R. S. Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination. Nature 317, 230–234 (1985).
Thomas, K. R., Folger, K. R. & Capecchi, M. R. High frequency targeting of genes to specific sites in the mammalian genome. Cell 44, 419–428 (1986).
Mansour, S. L., Thomas, K. R. & Capecchi, M. R. Disruption of the proto-oncogene Int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352 (1988).
Frese, K. K. & Tuveson, D. A. Maximizing mouse cancer models. Nat. Rev. Cancer 7, 645–658 (2007).
Rudin, N., Sugarman, E. & Haber, J. E. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics 122, 519–534 (1989).
Plessis, A., Perrin, A., Haber, J. E. & Dujon, B. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics 130, 451–460 (1992).
Rouet, P., Smih, F. & Jasin, M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc. Natl Acad. Sci. USA 91, 6064–6068 (1994).
Choulika, A., Perrin, A., Dujon, B. & Nicolas, J. F. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 1968–1973 (1995).
Bibikova, M., Golic, M., Golic, K. G. & Carroll, D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002).
Bibikova, M., Beumer, K., Trautman, J. K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).
Urnov, F. D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nat. Cell Biol. 435, 646–651 (2005).
Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).
Li, T. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39, 359–372 (2011).
Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).
Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).
Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).
Joung, J. K. & Sander, J. D. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14, 49–55 (2013).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).
Cho, S. W., Kim, S., Kim, J. M. & Kim, J.-S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–1121 (2013).
Hou, Z. et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl Acad. Sci. USA 110, 15644–15649 (2013).
Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014).
Vogelstein, B. et al. Cancer genome landscapes. Science 339, 1546–1558 (2013).
Denicola, G. M. et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 475, 106–109 (2011).
Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J.-S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).
Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).
Malina, A. et al. Repurposing CRISPR/Cas9 for in situ functional assays. Genes Dev. 27, 2602–2614 (2013).
Shalem, O. et al. Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
Heyer, J., Kwong, L. N., Lowe, S. W. & Chin, L. Non-germline genetically engineered mouse models for translational cancer research. Nat. Rev. Cancer 10, 470–480 (2010).
Engelman, J. A. et al. Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers. Nat. Med. 14, 1351–1356 (2008).
Chen, Z. et al. A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response. Nature 483, 613–617 (2012).
Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).
Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).
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).
Guerra, C. et al. Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 4, 111–120 (2003).
Findlay, G. M., Boyle, E. A., Hause, R. J., Klein, J. C. & Shendure, J. Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120–123 (2014).
Dow, L. E. & Lowe, S. W. Life in the fast lane: mammalian disease models in the genomics era. Cell 148, 1099–1109 (2012).
Chen, C. et al. MLL3 is a haploinsufficient 7q tumor suppressor in acute myeloid leukemia. Cancer Cell 25, 652–665 (2014).
Heckl, D. et al. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR–Cas9 genome editing. Nat. Biotechnol. 32, 941–946 (2014).
Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).
Jackson, E. et al. The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer Res. 65, 10280–10288 (2005).
Sanchez-Rivera, F. J. et al. Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature 516, 428–431 (2014).
Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423–427 (2014).
Soda, M. et al. Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer. Nature 448, 561–566 (2007).
Shaw, A. T. et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N. Engl. J. Med. 368, 2385–2394 (2013).
Blasco, R. B. et al. Simple and rapid in vivo generation of chromosomal rearrangements using CRISPR/Cas9 technology. Cell Rep. 9, 1219–1227 (2014).
Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).
Platt, R. J. et al. CRISPR–Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).
Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014).
Dow, L. E. et al. Inducible in vivo genome editing with CRISPR–Cas9. Nat. Biotechnol. 33, 390–394 (2015).
Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Whitworth, K. M. et al. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol. Reprod. 91, 78 (2014).
Niu, Y. et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156, 836–843 (2014).
Crystal, A. S. et al. Patient-derived models of acquired resistance can identify effective drug combinations for cancer. Science 346, 1480–1486 (2014).
Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).
Sadelain, M., Brentjens, R. & Rivière, I. The basic principles of chimeric antigen receptor design. Cancer Discov. 3, 388–398 (2013).
Sadelain, M., Papapetrou, E. P. & Bushman, F. D. Safe harbours for the integration of new DNA in the human genome. Nat. Rev. Cancer 12, 51–58 (2012).
Bikard, D. et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR–Cas system. Nucleic Acids Res. 41, 7429–7437 (2013).
Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).
Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR–Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).
Cheng, A. W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).
Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2014).
Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015).
Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 (2014).
Koike-Yusa, H., Li, Y., Tan, E.-P., Velasco-Herrera, M. D. C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2013).
Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014).
Chen, S. et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160, 1246–1260 (2015).
Matano, M. et al. Modeling colorectal cancer using CRISPR–Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).
Mansour, M. R. et al. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element. Science 346, 1373–1377 (2014).
Choi, P. S. & Meyerson, M. Targeted genomic rearrangements using CRISPR/Cas technology. Nat. Commun. 5, 3728 (2014).
Torres, R. et al. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system. Nat. Commun. 5, 3964 (2014).
Ghezraoui, H. et al. Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol. Cell 55, 829–842 (2014).
Xiao, A. et al. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 41, e141 (2013).
Canver, M. C. et al. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J. Biol. Chem. 289, 21312–21324 (2014).
Han, J. et al. Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9. RNA Biol. 11, 829–835 (2014).
Ho, T.-T. et al. Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines. Nucleic Acids Res. 43, e17 (2014).
Essletzbichler, P. et al. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res. 24, 2059–2065 (2014).
Work in the Jacks laboratory is supported by the Howard Hughes Medical Institute, the National Cancer Institute (US National Institutes of Health), the Ludwig Fund for Cancer Research, the Lustgarten Foundation and the US Department of Defense. T.J. is a Daniel K. Ludwig Scholar and the David H. Koch Professor of Biology at Massachusetts Institute of Technology.
The authors declare no competing financial interests.
Tumour tissues or cell lines from one species that have been implanted into a recipient of the same species.
- Cancer genes
Genes that have a causal role in carcinogenesis, such as tumour-promoting genes (commonly referred to as 'oncogenes') and tumour-suppressor genes.
- Chimeric antigen receptor-modified T cells
(CAR T cells). T cells that have been modified to express a synthetically engineered T cell receptor composed of a single-chain antibody, that allows for tumour recognition, fused with additional intracellular signalling domains derived from the T cell receptor and other co-stimulatory molecules.
- Conditional alleles
Engineered alleles, the expression of which is strictly dependent on the presence of a second component, such as Cre recombinase for loxP-based conditional alleles.
- CRISPR RNA
(crRNA). In natural CRISPR (clustered regularly interspaced short palindromic repeats) systems, CRISPR loci contain crRNAs that are composed of a repeat portion and a variable portion, the latter corresponding to invader-specific DNA sequences that form the basis of this prokaryotic adaptive immune system.
- Genetically engineered mouse models
(GEMMs). Mice genetically engineered to express either exogenous or endogenous mutated genes. For GEMMs of cancer, the modified genes are usually cancer genes.
A genetic state in which the presence of a single functional copy of a gene (as a result of mutation or loss of the other functional copy of the gene) produces a mutant phenotype.
- Homology-directed repair
(HDR). A precise DNA repair pathway in which the cell uses the homologous chromosome or sister chromatid (or an exogenous donor DNA molecule provided by the investigator) as a template for repairing a double-strand break. Homologous recombination is a form of HDR.
- 'Hotspot' regions
Specific regions of a gene in which mutations are observed with greater frequency.
- Hydrodynamic gene transfer
A technique that allows for delivery of DNA via the application of hydrodynamic pressure in capillaries, which greatly enhances endothelial and parenchymal cell permeability, leading to efficient transfer of DNA.
Mutations that arise because of small insertions or deletions of DNA sequences.
- Non-homologous end joining pathway
(NHEJ pathway). An error-prone cellular DNA repair pathway that involves the modification and subsequent ligation of two broken DNA ends generated by a double-strand break. This pathway can result in insertion or deletion mutations due to the addition or removal of nucleotides at the break before ligation.
- Organoid cultures
A type of in vitro culture in which stem cells or organ progenitors are embedded in a three-dimensional matrix in which they self-assemble into epithelia that very closely resemble the organ of origin. These organoid cultures offer several advantages over traditional cell culture, the main one being that they very closely recapitulate the physiology of the native epithelial organ of origin.
- Patient-derived xenografts
A type of xenograft in which fresh tumour tissue obtained from a cancer patient is implanted directly into an immunodeficient mouse.
- Protospacer adjacent motif
(PAM). A nucleotide sequence immediately adjacent to the target sequence that is critically required for CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas)-mediated recognition and cleavage. In natural CRISPR systems, the PAM sequence allows for self versus non-self discrimination by virtue of the absence of PAM sequences in endogenous CRISPR loci.
- Single guide RNA
(sgRNA). A chimeric RNA molecule generated by the fusion of a CRISPR (clustered regularly interspaced short palindromic repeats) RNA (crRNA) and a trans-activating crRNA (tracrRNA) that guides Cas9 (CRISPR-associated 9) to a specific genomic target via a unique guide RNA sequence present in the 5′ region of the crRNA.
- Site-specific recombinases
Enzymes (for example, Cre and flippase (Flp)) that can catalyse the recombination between specific pairs of inverted repeat sequences (for example, loxP sequences for Cre and Flp recombination target (FRT) sequences for Flp).
- Trans-activating crRNA
(tracrRNA). In natural CRISPR (clustered regularly interspaced short palindromic repeats) systems, the tracrRNA is a trans-encoded RNA molecule that is critical for the processing of pre-CRISPR RNA (pre-crRNA) loci into mature crRNAs.
- Transcription activator-like effector nucleases
(TALENs). Site-specific DNA endonucleases that are composed of specific DNA-binding domains from TALE proteins and the nuclease domain of the FokI restriction enzyme.
Tumour tissues or cell lines from one species that have been implanted into another species. In cancer research, this frequently entails the implantation of material from one species into an ectopic or orthotopic site of an immunodeficient mouse.
- Zinc-finger nucleases
(ZFNs). Site-specific DNA endonucleases that are composed of specific DNA-binding domains from zinc-finger proteins and the nuclease domain of the FokI restriction enzyme.
About this article
Cite this article
Sánchez-Rivera, F., Jacks, T. Applications of the CRISPR–Cas9 system in cancer biology. Nat Rev Cancer 15, 387–393 (2015). https://doi.org/10.1038/nrc3950
This article is cited by
Nature Reviews Clinical Oncology (2023)
An Insight into Modern Targeted Genome-Editing Technologies with a Special Focus on CRISPR/Cas9 and its Applications
Molecular Biotechnology (2023)
Improvement of Soybean; A Way Forward Transition from Genetic Engineering to New Plant Breeding Technologies
Molecular Biotechnology (2023)
CRISPR/Cas9-mediated LINC00511 knockout strategies, increased apoptosis of breast cancer cells via suppressing antiapoptotic genes
Biological Procedures Online (2022)
Monitoring autochthonous lung tumors induced by somatic CRISPR gene editing in mice using a secreted luciferase
Molecular Cancer (2022)