RESUMO
Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.
Assuntos
Neoplasias da Mama/genética , Sistemas CRISPR-Cas , Edição de Genes , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , MutaçãoRESUMO
Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a 'Gene On' (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes or enzymes, such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.
Assuntos
Edição de Genes , Genes Reporter , Animais , Sequência de Bases , Linhagem Celular Tumoral , Resistência Microbiana a Medicamentos , Células HEK293 , Humanos , Integrases/metabolismo , Camundongos , Células NIH 3T3 , Recombinação Genética/genéticaRESUMO
Base editing is a powerful technology that enables programmable conversion of single nucleotides in the mammalian genome. Base editors consist of a partially active Cas9 nuclease (Cas9D10A) tethered to a natural or synthetic DNA modifying enzyme. Though only recently described, BE has already shown enormous potential for basic and translational research, allowing the creation or repair of disease alleles in a variety of cell types and model organisms. In the past 2â¯years, a vast array of new and modified base editor variants have been described, expanding the flexibility and usefulness of the approach. Though simple in concept, effective implementation of base editing requires an understanding of the advantages and limitations of each of these tools. Here, we provide an overview of the concepts of DNA base editing, and discuss the recent progress toward the development of optimized base editing systems for mammalian cells. In addition, we highlight key technical aspects of designing and executing BE experiments, and provide detailed experimental examples of successful base editing in cell lines and organoids to help guide the effective use of these tools for genome modification.
Assuntos
Sequência de Bases/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , DNA/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
BACKGROUND: Eosinophils secrete several granules that are involved in the propagation of inflammatory responses in patients with pathologies such as asthma. OBJECTIVE: We hypothesized that some of these granules are exosomes, which, when transferred to the recipient cells, could modulate asthma progression. METHODS: Eosinophils were purified from peripheral blood and cultured with or without IFN-γ or eotaxin. Multivesicular bodies (MVBs) in eosinophils were studied by using fluorescence microscopy, transmission electron microscopy (TEM), and flow cytometry. Exosome secretion was measured and exosome characterization was performed with TEM, Western blotting, and NanoSight analysis. RESULTS: Generation of MVBs in eosinophils was confirmed by using fluorescence microscopy and flow cytometry and corroborated by means of TEM. Having established that eosinophils contain MVBs, our aim was to demonstrate that eosinophils secrete exosomes. To do this, we purified exosomes from culture medium of eosinophils and characterized them. Using Western blot analysis, we demonstrated that eosinophils secreted exosomes and that the discharge of exosomes to extracellular media increases after IFN-γ stimulation. We measured exosome size and quantified exosome production from healthy and asthmatic subjects using nanotracking analysis. We found that exosome production was augmented in asthmatic patients. CONCLUSION: Our findings are the first to demonstrate that eosinophils contain functional MVBs and secrete exosomes and that their secretion is increased in asthmatic patients. Thus exosomes might play an important role in the progression of asthma and eventually be considered a biomarker.
Assuntos
Asma/diagnóstico , Eosinófilos/metabolismo , Exossomos/metabolismo , Corpos Multivesiculares/metabolismo , Asma/imunologia , Asma/metabolismo , Asma/patologia , Biomarcadores/análise , Biomarcadores/metabolismo , Estudos de Casos e Controles , Fracionamento Celular , Separação Celular , Quimiocina CCL11/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/ultraestrutura , Exossomos/imunologia , Exossomos/ultraestrutura , Humanos , Interferon gama/farmacologia , Microscopia Eletrônica de Transmissão , Corpos Multivesiculares/imunologia , Corpos Multivesiculares/ultraestrutura , Tamanho das Organelas , Cultura Primária de CélulasRESUMO
Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.
Assuntos
Edição de Genes , Neoplasias , Camundongos , Animais , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , Neoplasias/genética , Neoplasias/terapia , PulmãoRESUMO
To better understand T lymphocyte costimulation by inducible costimulator (ICOS; H4; CD278), we analyzed proteins binding to ICOS peptides phosphorylated at the Y(191)MFM motif. Phosphorylated ICOS binds class IA phosphatidyl inositol 3-kinase (PI3-K) p85α, p50-55α and p85ß regulatory subunits and p110α, p110δ and p110ß catalytic subunits. Intriguingly, T cells expressed high levels of both p110α or p110δ catalytic subunits, yet ICOS peptides, cell surface ICOS or PI3-kinase class IA regulatory subunits preferentially coprecipitated p110α catalytic subunits. Silencing p110α or p110δ partially inhibited Akt/PKB activation induced by anti-CD3 plus anti-ICOS antibodies. However, silencing p110α enhanced and silencing p110δ inhibited Erk activation. Both p110α- and p110δ-specific inhibitors blocked cytokine secretion induced by TCR/CD3 activation with or without ICOS costimulus, but only p110α inhibitors blocked ICOS-induced cell elongation. Thus, p110α and p110δ are essential to optimal T cell activation, but their abundance and activity differentially tune up distinct ICOS signaling pathways.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Ativação Linfocitária/fisiologia , Fosfatidilinositol 3-Quinase/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Proteína Coestimuladora de Linfócitos T Induzíveis , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinase/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Asthma and nonasthmatic eosinophilic bronchitis (NAEB) are respiratory disorders characterized by a predominance of Th2 cells and eosinophilic inflammation. Suppressors of cytokine signaling (SOCS) proteins play an important role in Th2-mediated allergic responses through control of the balance between Th1 and Th2 cells, particularly, SOCS3 and SOCS5. The aim of this study was to analyze SOCS expression in human peripheral blood eosinophils from patients with asthma, NAEB and healthy controls. SOCS expression in eosinophils from subjects was demonstrated by different techniques. Results showed that expression of SOCS3 in eosinophils and CD4 T cells from patients was higher than in healthy subjects. In addition, we demonstrated that prostaglandin E2 (PGE2) and Th2 cytokines are able to upregulate SOCS3 production in eosinophils and attenuate its degranulation. In conclusion, eosinophils are able to transcribe and translate SOCS3 protein and can contribute to the regulation of the Th1/Th2 balance through SOCS3 production.
Assuntos
Asma/sangue , Bronquite/sangue , Eosinofilia/sangue , Eosinófilos/metabolismo , Transdução de Sinais/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Equilíbrio Th1-Th2 , Adulto , Asma/genética , Asma/imunologia , Asma/patologia , Bronquite/genética , Bronquite/imunologia , Bronquite/patologia , Broncoscopia , Estudos de Casos e Controles , Separação Celular , Células Cultivadas , Dinoprostona/análise , Dinoprostona/biossíntese , Eosinofilia/genética , Eosinofilia/imunologia , Eosinofilia/patologia , Eosinófilos/imunologia , Feminino , Expressão Gênica , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Transdução de Sinais/genética , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
In this issue of Cancer Discovery, Lo and colleagues use CRISPR-based genome engineering in primary human gastric organoids to reveal the functional consequences of ARID1A loss in the early stages of gastric cancer. They show that ARID1A disruption is not tolerated in wild-type organoids, but in the context of TP53 loss, leads to WNT suppression, mucinous metaplasia, enhanced tumorigenicity, and selectively toxicity to BIRC5/Survivin inhibition.See related article by Lo et al., p. 1562.
Assuntos
Neoplasias Gástricas , Proteínas de Ligação a DNA , Humanos , Neoplasias Gástricas/genética , Fatores de Transcrição/genéticaRESUMO
The WNT pathway is a fundamental regulator of intestinal homeostasis, and hyperactivation of WNT signaling is the major oncogenic driver in colorectal cancer. To date, there are no described mechanisms that bypass WNT dependence in intestinal tumors. Here, we show that although WNT suppression blocks tumor growth in most organoid and in vivo colorectal cancer models, the accumulation of colorectal cancer-associated genetic alterations enables drug resistance and WNT-independent growth. In intestinal epithelial cells harboring mutations in KRAS or BRAF, together with disruption of TP53 and SMAD4, transient TGFß exposure drives YAP/TAZ-dependent transcriptional reprogramming and lineage reversion. Acquisition of embryonic intestinal identity is accompanied by a permanent loss of adult intestinal lineages, and long-term WNT-independent growth. This work identifies genetic and microenvironmental factors that drive WNT inhibitor resistance, defines a new mechanism for WNT-independent colorectal cancer growth, and reveals how integration of associated genetic alterations and extracellular signals can overcome lineage-dependent oncogenic programs. SIGNIFICANCE: Colorectal and intestinal cancers are driven by mutations in the WNT pathway, and drugs aimed at suppressing WNT signaling are in active clinical development. Our study identifies a mechanism of acquired resistance to WNT inhibition and highlights a potential strategy to target those drug-resistant cells.This article is highlighted in the In This Issue feature, p. 1426.
Assuntos
Neoplasias Intestinais/genética , Via de Sinalização Wnt/genética , Animais , Linhagem Celular Tumoral , Humanos , CamundongosRESUMO
KRAS is the most frequently mutated oncogene in cancer, yet there is little understanding of how specific KRAS amino acid changes affect tumor initiation, progression, or therapy response. Using high-fidelity CRISPR-based engineering, we created an allelic series of new LSL-Kras mutant mice, reflecting codon 12 and 13 mutations that are highly prevalent in lung (KRASG12C), pancreas (KRASG12R), and colon (KRASG13D) cancers. Induction of each allele in either the murine colon or pancreas revealed striking quantitative and qualitative differences between KRAS mutants in driving the early stages of transformation. Furthermore, using pancreatic organoid models, we show that KRASG13D mutants are sensitive to EGFR inhibition, whereas KRASG12C-mutant organoids are selectively responsive to covalent G12C inhibitors only when EGFR is suppressed. Together, these new mouse strains provide an ideal platform for investigating KRAS biology in vivo and for developing preclinical precision oncology models of KRAS-mutant pancreas, colon, and lung cancers. SIGNIFICANCE: KRAS is the most frequently mutated oncogene. Here, we describe new preclinical models that mimic tissue-selective KRAS mutations and show that each mutation has distinct cellular consequences in vivo and carries differential sensitivity to targeted therapeutic agents.See related commentary by Kostyrko and Sweet-Cordero, p. 1626.This article is highlighted in the In This Issue feature, p. 1611.
Assuntos
Alelos , Oncogenes/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular Tumoral , Humanos , FenótipoRESUMO
In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.
Assuntos
Adenina/metabolismo , Edição de Genes/métodos , Tirosinemias/genética , Animais , Modelos Animais de Doenças , Feminino , Células HEK293 , Hepatócitos/metabolismo , Humanos , Hidrolases/genética , Fígado/metabolismo , Mutação Puntual , RNA/administração & dosagemRESUMO
The majority of colorectal cancers show hyperactivated WNT signaling due to inactivating mutations in the adenomatous polyposis coli (APC) tumor suppressor. Genetically restoring APC suppresses WNT and induces rapid and sustained tumor regression, implying that reengaging this endogenous tumor-suppressive mechanism may be an effective therapeutic strategy. Here, using new animal models, human cell lines, and ex vivo organoid cultures, we show that tankyrase (TNKS) inhibition can control WNT hyperactivation and provide long-term tumor control in vivo, but that effective responses are critically dependent on how APC is disrupted. Mutant APC proteins truncated within the mutation cluster region physically engage the destruction complex and suppress the WNT transcriptional program, while APC variants with early truncations (e.g., Apc Min) show limited interaction with AXIN1 and ß-catenin, and do not respond to TNKS blockade. Together, this work shows that TNKS inhibition, like APC restoration, can reestablish endogenous control of WNT/ß-catenin signaling, but that APC genotype is a crucial determinant of this response. SIGNIFICANCE: This study reveals how subtle changes to the mutations in a critical colorectal tumor suppressor, APC, influence the cellular response to a targeted therapy. It underscores how investigating the specific genetic alterations that occur in human cancer can identify important biological mechanisms of drug response and resistance.This article is highlighted in the In This Issue feature, p. 1325.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Mutação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Tanquirases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Marcação de Genes , Humanos , Camundongos , Terapia de Alvo Molecular , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Interferência de RNA , Tanquirases/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Pustulose Exantematosa Aguda Generalizada/induzido quimicamente , Analgésicos não Narcóticos/efeitos adversos , Anti-Inflamatórios não Esteroides/efeitos adversos , Toxidermias/etiologia , Ibuprofeno/efeitos adversos , Pustulose Exantematosa Aguda Generalizada/imunologia , Basófilos/imunologia , Toxidermias/imunologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
CRISPR base editing enables the creation of targeted single-base conversions without generating double-stranded breaks. However, the efficiency of current base editors is very low in many cell types. We reengineered the sequences of BE3, BE4Gam, and xBE3 by codon optimization and incorporation of additional nuclear-localization sequences. Our collection of optimized constitutive and inducible base-editing vector systems dramatically improves the efficiency by which single-nucleotide variants can be created. The reengineered base editors enable target modification in a wide range of mouse and human cell lines, and intestinal organoids. We also show that the optimized base editors mediate efficient in vivo somatic editing in the liver in adult mice.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Linhagem Celular , Variação Genética , Humanos , CamundongosRESUMO
Defining the genetic drivers of cancer progression is a key in understanding disease biology and developing effective targeted therapies. Chromosome rearrangements are a common feature of human malignancies, but whether they represent bona fide cancer drivers and therapeutically actionable targets, requires functional testing. Here, we describe the generation of transgenic, inducible CRISPR-based mouse systems to engineer and study recurrent colon cancer-associated EIF3E-RSPO2 and PTPRK-RSPO3 chromosome rearrangements in vivo. We show that both Rspo2 and Rspo3 fusion events are sufficient to initiate hyperplasia and tumour development in vivo, without additional cooperating genetic events. Rspo-fusion tumours are entirely Wnt-dependent, as treatment with an inhibitor of Wnt secretion, LGK974, drives rapid tumour clearance from the intestinal mucosa without effects on normal intestinal crypts. Altogether, our study provides direct evidence that endogenous Rspo2 and Rspo3 chromosome rearrangements can initiate and maintain tumour development, and indicate a viable therapeutic window for LGK974 treatment of RSPO-fusion cancers.
Assuntos
Aberrações Cromossômicas , Cromossomos/genética , Neoplasias do Colo/genética , Rearranjo Gênico , Trombospondinas/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Humanos , Intestinos/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pirazinas/administração & dosagem , Piridinas/administração & dosagem , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Generating new mouse models of cancer is a challenging and laborious task that can require years of investment to interrogate a single gene. Now, Jonkers and colleagues describe the first application of CRISPR-based genome editing in the mammary gland, providing a new approach to dissect complex genetic interactions in vivo.