MOLECULAR & CELLULAR ONCOLOGY 2016, VOL. 3, NO. 6, e1227894 (3 pages) http://dx.doi.org/10.1080/23723556.2016.1227894
COMMENTARY
Modeling intratumor heterogeneity through CRISPR-barcodes Alexis Guerneta,b, Stuart A. Aaronsonc, Youssef Anouara,b, and Luca Grumolato
a,b
a Normandie Univ, UNIROUEN, INSERM, DC2N, Rouen, France; bInstitute for Research and Innovation in Biomedicine, Rouen, France; cDepartment of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
ABSTRACT
ARTICLE HISTORY
We have devised a barcoding strategy to recapitulate cancer evolution through the emergence of subclonal mutations of interest, whose effects can be monitored in a dynamic manner. This approach can be easily adapted for a variety of applications, including combined modeling of multiple mechanisms of drug resistance or repair of oncogenic driver mutations in addicted cancer cells.
Received 17 August 2016 Revised 18 August 2016 Accepted 19 August 2016 KEYWORDS
ALK; APC; CRISPR/Cas9; EGFR; genetic barcoding; non-small cell lung cancer; resistance; TP53; tumor heterogeneity
Cancer is an evolutionary process in which the stepwise accumulation of genetic alterations is shaped by Darwinian selection. As a result, each tumor is composed of a complex mixture of clonal cell subpopulations containing a partially overlapping, but distinct, pattern of driver and passenger mutations. Such intratumor heterogeneity has dramatic consequences, not only for cancer progression and metastatic spread, but also for resistance to therapy.1,2 While snapshots of the clonal architecture of a particular tumor at a given stage can now be obtained through deep sequencing and mathematical modeling, such complexity is generally not taken into account when investigating the effects of a particular oncogenic mutation. We recently devised a novel approach based on new technologies for DNA editing to recapitulate and trace the emergence of a new mutation in a subset of cancer cells, thus enabling functional studies on a gene of interest in a context that mimics intratumor heterogeneity.3 Originally an adaptive immune system in prokaryotes, CRISPR (clustered regularly interspaced short palindromic repeats) has been engineered into a new powerful tool for genome editing.4,5 This system is composed of the Cas9 nuclease from S. Pyogenes and a short RNA sequence, the singleguide RNA (sgRNA). When co-expressed in cells, Cas9 and the sgRNA form a complex that specifically recognizes a particular DNA sequence through Watson-Crick pairing and promotes its cleavage. The double-strand DNA break induced by CRISPR/ Cas9 can trigger two distinct cellular mechanisms for DNA repair that can be exploited for DNA editing: error-prone nonhomologous end-joining (NHEJ) and high-fidelity homologydirected repair (HDR). DNA repair through NHEJ frequently generates insertions or deletions (indels), which can alter the frame of a coding sequence and result in gene inactivation. In HDR, a donor DNA co-introduced into the cells functions as a template for precise repair; through appropriate design of the donor DNA this mechanism can be used to generate a wide
CONTACT Luca Grumolato © 2016 Taylor & Francis Group, LLC
[email protected]
range of genetic modifications, including specific point mutations or the insertion of an entire gene. Depending on the extent of the desired modification, a single-stranded DNA oligonucleotide (ssODN) or a double-stranded DNA targeting construct can be used as donor DNA for HDR.4,5 Despite the undeniable potential of this new technology, two major intrinsic limitations must be considered when applying CRISPR/Cas9. First, in certain contexts this system can tolerate a few mismatches between the sgRNA and its target sequence, which can result in off-target DNA cleavage.4-7 The second limitation is related to the fact that the efficiency of CRISPR/Cas9mediated DNA editing can vary substantially depending on the cell model, the sgRNA, and the targeted DNA. Thus, in a given experiment only a fraction of cells within a population will contain the desired genetic modification. Hence, derivation and subsequent analysis of a certain number of individual clones constitute an almost inevitable step8 that is obviously not compatible with modifications that have a negative impact on cell growth. We used CRISPR/Cas9 technology to insert a potentially functional modification in the sequence of a gene of interest coupled to a series of silent point mutations, serving as a genetic label for cell tracing. Because of the low efficiency of HDR, within a mass population the modified allele would be introduced only in a small fraction of tumor cells, thus mimicking the emergence of a new mutant clone. In parallel, a distinct subpopulation was tagged with a second barcode consisting of different silent mutations, which provided an internal control for possible CRISPR off-target effects. After exposing the cells to a given selective condition, we assessed the effects of the mutation of interest by measuring the proportion of each barcode in genomic DNA by real-time quantitative PCR or deep sequencing (Fig. 1). We applied this strategy to recapitulate different mechanisms of resistance to inhibitors of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer
e1227894-2
A. GUERNET ET AL.
Figure 1. CRISPR-barcoding strategy. Cells are co-transfected with a vector for Cas9 and sgRNA expression, together with a single-stranded DNA oligonucleotide (ssODN) containing the mutation of interest (Mutant) coupled with a series of silent mutations. In parallel, a ssODN containing only silent mutations (WT) is used as an internal control for off-target DNA cleavage by Cas9. The cells are then exposed to a given selective condition and the effects of the mutation on cell growth, invasion, signaling activation, or tumorigenicity can be assessed by quantitative PCR (qPCR) or deep sequencing. A similar approach was used to introduce highly complex barcodes through a degenerate ssODN in the different clones that constitute a mass population of cancer cells. We used this strategy to compare the relative fitness of breast cancer cells to grow in vivo after injection in immunodeficient mice and to investigate the effects of the EGFR inhibitor gefitinib in lung cancer cells.
cells, and generated a multiplex model for in vitro and in vivo investigation of new therapeutic strategies to prevent or delay propagation of resistant cells. Using CRISPR-barcoding, we also manipulated the endogenous sequence of different oncogenes and tumor suppressors, including adenomatous polyposis coli (APC), tumor protein p53 (TP53) and anaplastic lymphoma kinase (ALK), and showed that this approach can be used to inactivate a gene through insertion of a STOP codon or to repair oncogenic mutations in addicted cancer cells. Finally, through insertion of a degenerate sequence at a specific genome location, we simultaneously traced the fate of several thousand different clones within a mass population of cancer cells upon inoculation in immunodeficient mice or in the presence of a therapeutic agent (Fig. 1), demonstrating that CRISPR-barcoding can represent a convenient alternative
strategy of randomly integrating lentiviral libraries9,10 to investigate intratumor heterogeneity. In conclusion, our study establishes a novel and unique means to recapitulate genetic heterogeneity in virtually all types of cancer cells that can be easily implemented to assess effects on cell growth/survival, invasion, transcriptional activity, resistance/sensitivity to a particular agent, and tumorigenicity. Compared with other CRISPR/Cas9-based approaches, our strategy provides several major advantages, including (i) the ability to investigate genome modifications that negatively affect cell growth; (ii) an internal control for potential off-target cleavage by the sgRNA-Cas9 complex; (iii) the use of a cell mass-population, thus preventing potential bias due to clonal selection; (iv) the fact that the system does not require high efficiency of DNA editing, which substantially broadens the choice
MOLECULAR & CELLULAR ONCOLOGY
of cell models that can be used; (v) ease of multiplexing; (vi) better cost- and time-effectiveness.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding This work was supported by the Institut National de la Sante et de la Recherche Medicale (INSERM), Universite de Rouen Normandie, Ligue contre le Cancer de Haute Normandie, the National Cancer Institute (P01CA080058), and the Breast Cancer Research Foundation. A.G is recipient of a doctoral fellowship from the Normandy Region. L.G. was supported by a Chair of Excellence program from INSERM and Universite de Rouen Normandie.
ORCID Luca Grumolato
http://orcid.org/0000-0001-8231-3032
References 1. Kleppe M, Levine RL. Tumor heterogeneity confounds and illuminates: assessing the implications. Nat Med 2014; 20:342-4; PMID:24710377; http://dx.doi.org/10.1038/nm.3522 2. McGranahan N, Swanton C. Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell 2015; 27:15-26; PMID:25584892; http://dx.doi.org/10.1016/j.ccell.2014.12.001
e1227894-3
3. Guernet A, Mungamuri SK, Cartier D, Sachidanandam R, Jayaprakash A, Adriouch S, Vezain M, Charbonnier F, Rohkin G, Coutant S, et al. CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations. Mol Cell 2016; 63:526-38; PMID:27453044; http://dx.doi.org/10.1016/j. molcel.2016.06.017 4. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 2014; 346:1258096; PMID:25430774; http://dx.doi.org/10.1126/science.1258096 5. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157:1262-78; PMID:24906146; http://dx.doi.org/10.1016/j.cell.2014.05.010 6. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013; 31:822-6; PMID:23792628; http://dx.doi.org/10.1038/nbt.2623 7. Kuscu C, Arslan S, Singh R, Thorpe J, Adli M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Bio 2014; 32:677-83; PMID:24837660; http://dx.doi.org/ 10.1038/nbt.2916 8. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protocols 2013; 8:2281-308; PMID:24157548; http://dx.doi.org/10.1038/nprot.2013.143 9. Bhang HE, Ruddy DA, Krishnamurthy Radhakrishna V, Caushi JX, Zhao R, Hims MM, Singh AP, Kao I, Rakiec D, Shaw P, et al. Studying clonal dynamics in response to cancer therapy using high-complexity barcoding. Nat Med 2015; 21:440-8; PMID:25849130; http://dx.doi. org/10.1038/nm.3841 10. Nguyen LV, Pellacani D, Lefort S, Kannan N, Osako T, Makarem M, Cox CL, Kennedy W, Beer P, Carles A, et al. Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells. Nature 2015; 528:267-71
COMMENTARY
Modeling intratumor heterogeneity through CRISPR-barcodes Alexis Guerneta,b, Stuart A. Aaronsonc, Youssef Anouara,b, and Luca Grumolato
a,b
a Normandie Univ, UNIROUEN, INSERM, DC2N, Rouen, France; bInstitute for Research and Innovation in Biomedicine, Rouen, France; cDepartment of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
ABSTRACT
ARTICLE HISTORY
We have devised a barcoding strategy to recapitulate cancer evolution through the emergence of subclonal mutations of interest, whose effects can be monitored in a dynamic manner. This approach can be easily adapted for a variety of applications, including combined modeling of multiple mechanisms of drug resistance or repair of oncogenic driver mutations in addicted cancer cells.
Received 17 August 2016 Revised 18 August 2016 Accepted 19 August 2016 KEYWORDS
ALK; APC; CRISPR/Cas9; EGFR; genetic barcoding; non-small cell lung cancer; resistance; TP53; tumor heterogeneity
Cancer is an evolutionary process in which the stepwise accumulation of genetic alterations is shaped by Darwinian selection. As a result, each tumor is composed of a complex mixture of clonal cell subpopulations containing a partially overlapping, but distinct, pattern of driver and passenger mutations. Such intratumor heterogeneity has dramatic consequences, not only for cancer progression and metastatic spread, but also for resistance to therapy.1,2 While snapshots of the clonal architecture of a particular tumor at a given stage can now be obtained through deep sequencing and mathematical modeling, such complexity is generally not taken into account when investigating the effects of a particular oncogenic mutation. We recently devised a novel approach based on new technologies for DNA editing to recapitulate and trace the emergence of a new mutation in a subset of cancer cells, thus enabling functional studies on a gene of interest in a context that mimics intratumor heterogeneity.3 Originally an adaptive immune system in prokaryotes, CRISPR (clustered regularly interspaced short palindromic repeats) has been engineered into a new powerful tool for genome editing.4,5 This system is composed of the Cas9 nuclease from S. Pyogenes and a short RNA sequence, the singleguide RNA (sgRNA). When co-expressed in cells, Cas9 and the sgRNA form a complex that specifically recognizes a particular DNA sequence through Watson-Crick pairing and promotes its cleavage. The double-strand DNA break induced by CRISPR/ Cas9 can trigger two distinct cellular mechanisms for DNA repair that can be exploited for DNA editing: error-prone nonhomologous end-joining (NHEJ) and high-fidelity homologydirected repair (HDR). DNA repair through NHEJ frequently generates insertions or deletions (indels), which can alter the frame of a coding sequence and result in gene inactivation. In HDR, a donor DNA co-introduced into the cells functions as a template for precise repair; through appropriate design of the donor DNA this mechanism can be used to generate a wide
CONTACT Luca Grumolato © 2016 Taylor & Francis Group, LLC
[email protected]
range of genetic modifications, including specific point mutations or the insertion of an entire gene. Depending on the extent of the desired modification, a single-stranded DNA oligonucleotide (ssODN) or a double-stranded DNA targeting construct can be used as donor DNA for HDR.4,5 Despite the undeniable potential of this new technology, two major intrinsic limitations must be considered when applying CRISPR/Cas9. First, in certain contexts this system can tolerate a few mismatches between the sgRNA and its target sequence, which can result in off-target DNA cleavage.4-7 The second limitation is related to the fact that the efficiency of CRISPR/Cas9mediated DNA editing can vary substantially depending on the cell model, the sgRNA, and the targeted DNA. Thus, in a given experiment only a fraction of cells within a population will contain the desired genetic modification. Hence, derivation and subsequent analysis of a certain number of individual clones constitute an almost inevitable step8 that is obviously not compatible with modifications that have a negative impact on cell growth. We used CRISPR/Cas9 technology to insert a potentially functional modification in the sequence of a gene of interest coupled to a series of silent point mutations, serving as a genetic label for cell tracing. Because of the low efficiency of HDR, within a mass population the modified allele would be introduced only in a small fraction of tumor cells, thus mimicking the emergence of a new mutant clone. In parallel, a distinct subpopulation was tagged with a second barcode consisting of different silent mutations, which provided an internal control for possible CRISPR off-target effects. After exposing the cells to a given selective condition, we assessed the effects of the mutation of interest by measuring the proportion of each barcode in genomic DNA by real-time quantitative PCR or deep sequencing (Fig. 1). We applied this strategy to recapitulate different mechanisms of resistance to inhibitors of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer
e1227894-2
A. GUERNET ET AL.
Figure 1. CRISPR-barcoding strategy. Cells are co-transfected with a vector for Cas9 and sgRNA expression, together with a single-stranded DNA oligonucleotide (ssODN) containing the mutation of interest (Mutant) coupled with a series of silent mutations. In parallel, a ssODN containing only silent mutations (WT) is used as an internal control for off-target DNA cleavage by Cas9. The cells are then exposed to a given selective condition and the effects of the mutation on cell growth, invasion, signaling activation, or tumorigenicity can be assessed by quantitative PCR (qPCR) or deep sequencing. A similar approach was used to introduce highly complex barcodes through a degenerate ssODN in the different clones that constitute a mass population of cancer cells. We used this strategy to compare the relative fitness of breast cancer cells to grow in vivo after injection in immunodeficient mice and to investigate the effects of the EGFR inhibitor gefitinib in lung cancer cells.
cells, and generated a multiplex model for in vitro and in vivo investigation of new therapeutic strategies to prevent or delay propagation of resistant cells. Using CRISPR-barcoding, we also manipulated the endogenous sequence of different oncogenes and tumor suppressors, including adenomatous polyposis coli (APC), tumor protein p53 (TP53) and anaplastic lymphoma kinase (ALK), and showed that this approach can be used to inactivate a gene through insertion of a STOP codon or to repair oncogenic mutations in addicted cancer cells. Finally, through insertion of a degenerate sequence at a specific genome location, we simultaneously traced the fate of several thousand different clones within a mass population of cancer cells upon inoculation in immunodeficient mice or in the presence of a therapeutic agent (Fig. 1), demonstrating that CRISPR-barcoding can represent a convenient alternative
strategy of randomly integrating lentiviral libraries9,10 to investigate intratumor heterogeneity. In conclusion, our study establishes a novel and unique means to recapitulate genetic heterogeneity in virtually all types of cancer cells that can be easily implemented to assess effects on cell growth/survival, invasion, transcriptional activity, resistance/sensitivity to a particular agent, and tumorigenicity. Compared with other CRISPR/Cas9-based approaches, our strategy provides several major advantages, including (i) the ability to investigate genome modifications that negatively affect cell growth; (ii) an internal control for potential off-target cleavage by the sgRNA-Cas9 complex; (iii) the use of a cell mass-population, thus preventing potential bias due to clonal selection; (iv) the fact that the system does not require high efficiency of DNA editing, which substantially broadens the choice
MOLECULAR & CELLULAR ONCOLOGY
of cell models that can be used; (v) ease of multiplexing; (vi) better cost- and time-effectiveness.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding This work was supported by the Institut National de la Sante et de la Recherche Medicale (INSERM), Universite de Rouen Normandie, Ligue contre le Cancer de Haute Normandie, the National Cancer Institute (P01CA080058), and the Breast Cancer Research Foundation. A.G is recipient of a doctoral fellowship from the Normandy Region. L.G. was supported by a Chair of Excellence program from INSERM and Universite de Rouen Normandie.
ORCID Luca Grumolato
http://orcid.org/0000-0001-8231-3032
References 1. Kleppe M, Levine RL. Tumor heterogeneity confounds and illuminates: assessing the implications. Nat Med 2014; 20:342-4; PMID:24710377; http://dx.doi.org/10.1038/nm.3522 2. McGranahan N, Swanton C. Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell 2015; 27:15-26; PMID:25584892; http://dx.doi.org/10.1016/j.ccell.2014.12.001
e1227894-3
3. Guernet A, Mungamuri SK, Cartier D, Sachidanandam R, Jayaprakash A, Adriouch S, Vezain M, Charbonnier F, Rohkin G, Coutant S, et al. CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations. Mol Cell 2016; 63:526-38; PMID:27453044; http://dx.doi.org/10.1016/j. molcel.2016.06.017 4. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 2014; 346:1258096; PMID:25430774; http://dx.doi.org/10.1126/science.1258096 5. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157:1262-78; PMID:24906146; http://dx.doi.org/10.1016/j.cell.2014.05.010 6. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013; 31:822-6; PMID:23792628; http://dx.doi.org/10.1038/nbt.2623 7. Kuscu C, Arslan S, Singh R, Thorpe J, Adli M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Bio 2014; 32:677-83; PMID:24837660; http://dx.doi.org/ 10.1038/nbt.2916 8. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protocols 2013; 8:2281-308; PMID:24157548; http://dx.doi.org/10.1038/nprot.2013.143 9. Bhang HE, Ruddy DA, Krishnamurthy Radhakrishna V, Caushi JX, Zhao R, Hims MM, Singh AP, Kao I, Rakiec D, Shaw P, et al. Studying clonal dynamics in response to cancer therapy using high-complexity barcoding. Nat Med 2015; 21:440-8; PMID:25849130; http://dx.doi. org/10.1038/nm.3841 10. Nguyen LV, Pellacani D, Lefort S, Kannan N, Osako T, Makarem M, Cox CL, Kennedy W, Beer P, Carles A, et al. Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells. Nature 2015; 528:267-71
