Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 32
Filtrar
Más filtros











Base de datos
Intervalo de año de publicación
1.
Nature ; 2024 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-39261725

RESUMEN

Layer 5 extratelencephalic (ET) neurons are present across neocortical areas and send axons to multiple subcortical targets1-6. Two cardinal subtypes exist7,8: (1) Slco2a1-expressing neurons (ETdist), which predominate in the motor cortex and project distally to the pons, medulla and spinal cord; and (2) Nprs1- or Hpgd-expressing neurons (ETprox), which predominate in the visual cortex and project more proximally to the pons and thalamus. An understanding of how area-specific ETdist and ETprox emerge during development is important because they are critical for fine motor skills and are susceptible to spinal cord injury and amyotrophic lateral sclerosis9-12. Here, using cross-areal mapping of axonal projections in the mouse neocortex, we identify the subtype-specific developmental dynamics of ET neurons. Whereas subsets of ETprox emerge by pruning of ETdist axons, others emerge de novo. We outline corresponding subtype-specific developmental transcriptional programs using single-nucleus sequencing. Leveraging these findings, we use postnatal in vivo knockdown of subtype-specific transcription factors to reprogram ET neuron connectivity towards more proximal targets. Together, these results show the functional transcriptional programs driving ET neuron diversity and uncover cell subtype-specific gene regulatory networks that can be manipulated to direct target specificity in motor corticofugal pathways.

2.
Nat Biomed Eng ; 2024 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-39322719

RESUMEN

Bispecific antibodies (biAbs) used in cancer immunotherapies rely on functional autologous T cells, which are often damaged and depleted in patients with haematological malignancies and in other immunocompromised patients. The adoptive transfer of allogeneic T cells from healthy donors can enhance the efficacy of biAbs, but donor T cells binding to host-cell antigens cause an unwanted alloreactive response. Here we show that allogeneic T cells engineered with a T-cell receptor that does not convert antigen binding into cluster of differentiation 3 (CD3) signalling decouples antigen-mediated T-cell activation from T-cell cytotoxicity while preserving the surface expression of the T-cell-receptor-CD3 signalling complex as well as biAb-mediated CD3 signalling and T-cell activation. In mice with CD19+ tumour xenografts, treatment with the engineered human cells in combination with blinatumomab (a clinically approved biAb) led to the recognition and clearance of tumour cells in the absence of detectable alloreactivity. Our findings support the development of immunotherapies combining biAbs and 'off-the-shelf' allogeneic T cells.

3.
Nature ; 632(8024): 411-418, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39048831

RESUMEN

It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.


Asunto(s)
Sistemas CRISPR-Cas , Hepatocitos , Neoplasias Hepáticas , Hígado , Metástasis de la Neoplasia , Proteínas del Tejido Nervioso , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Elementos Transponibles de ADN , Fluorescencia , Hepatocitos/metabolismo , Hepatocitos/citología , Hepatocitos/patología , Factor 4 Similar a Kruppel/metabolismo , Hígado/citología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Melanoma/metabolismo , Melanoma/patología , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Semaforinas/antagonistas & inhibidores , Semaforinas/metabolismo
4.
Nature ; 622(7982): 367-375, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37730998

RESUMEN

The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype-phenotype relationships in complex tissues in a high-throughput manner1,2. Here we introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening, termed AAV-Perturb-seq, a tuneable and broadly applicable method for transcriptional linkage analysis as well as high-throughput and high-resolution phenotyping of genetic perturbations in vivo. We applied AAV-Perturb-seq using gene editing and transcriptional inhibition to systematically dissect the phenotypic landscape underlying 22q11.2 deletion syndrome3,4 genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and previously undescribed pathways orchestrating neuronal functions in vivo that explain approximately 40% of the transcriptional changes observed in a 22q11.2-deletion mouse model. Our findings suggest that the 22q11.2-deletion syndrome transcriptional phenotype found in mature neurons may in part be due to the broad dysregulation of a class of genes associated with disease susceptibility that are important for dysfunctional RNA processing and synaptic function. Our study establishes a flexible and scalable direct in vivo method to facilitate causal understanding of biological and disease mechanisms with potential applications to identify genetic interventions and therapeutic targets for treating disease.


Asunto(s)
Sistemas CRISPR-Cas , Dependovirus , Edición Génica , Estudios de Asociación Genética , Análisis de la Célula Individual , Transcripción Genética , Animales , Humanos , Ratones , Dependovirus/genética , Estudios de Asociación Genética/métodos , Neuronas/metabolismo , Fenotipo , Corteza Prefrontal/metabolismo , Transcripción Genética/genética , Análisis de la Célula Individual/métodos , Sistemas CRISPR-Cas/genética , Síndrome de DiGeorge/tratamiento farmacológico , Síndrome de DiGeorge/genética , Modelos Animales de Enfermedad , Procesamiento Postranscripcional del ARN , Sinapsis/patología , Predisposición Genética a la Enfermedad
5.
Nat Commun ; 14(1): 6116, 2023 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-37777530

RESUMEN

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene deletions in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Ratones , Animales , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Eliminación de Gen , ARN Guía de Sistemas CRISPR-Cas , Ratones Noqueados , Fibras Musculares Esqueléticas
6.
Neuron ; 111(15): 2282-2311, 2023 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-37201524

RESUMEN

Genome engineering technologies provide an entry point into understanding and controlling the function of genetic elements in health and disease. The discovery and development of the microbial defense system CRISPR-Cas yielded a treasure trove of genome engineering technologies and revolutionized the biomedical sciences. Comprising diverse RNA-guided enzymes and effector proteins that evolved or were engineered to manipulate nucleic acids and cellular processes, the CRISPR toolbox provides precise control over biology. Virtually all biological systems are amenable to genome engineering-from cancer cells to the brains of model organisms to human patients-galvanizing research and innovation and giving rise to fundamental insights into health and powerful strategies for detecting and correcting disease. In the field of neuroscience, these tools are being leveraged across a wide range of applications, including engineering traditional and non-traditional transgenic animal models, modeling disease, testing genomic therapies, unbiased screening, programming cell states, and recording cellular lineages and other biological processes. In this primer, we describe the development and applications of CRISPR technologies while highlighting outstanding limitations and opportunities.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Animales , Humanos , Sistemas CRISPR-Cas/genética , Genoma , Genómica , ARN
7.
Cell Host Microbe ; 31(2): 164-166, 2023 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-36758514

RESUMEN

Engineered microbes show potential for diagnosing and treating diseases. In this issue of Cell Host & Microbe, Zou et al. develop an "intelligent" bacterial strain that detects and monitors an inflammation biomarker in the gut and responds by releasing an immunomodulator, thereby combining diagnosis and therapy for intestinal inflammation.


Asunto(s)
Bacterias , Inflamación , Humanos , Inflamación/diagnóstico
8.
Science ; 376(6594): eabm6038, 2022 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-35549411

RESUMEN

Transcriptional recording by CRISPR spacer acquisition from RNA endows engineered Escherichia coli with synthetic memory, which through Record-seq reveals transcriptome-scale records. Microbial sentinels that traverse the gastrointestinal tract capture a wide range of genes and pathways that describe interactions with the host, including quantitative shifts in the molecular environment that result from alterations in the host diet, induced inflammation, and microbiome complexity. We demonstrate multiplexed recording using barcoded CRISPR arrays, enabling the reconstruction of transcriptional histories of isogenic bacterial strains in vivo. Record-seq therefore provides a scalable, noninvasive platform for interrogating intestinal and microbial physiology throughout the length of the intestine without manipulations to host physiology and can determine how single microbial genetic differences alter the way in which the microbe adapts to the host intestinal environment.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Escherichia coli , Microbioma Gastrointestinal , Tracto Gastrointestinal , Interacciones Microbiota-Huesped , Animales , Escherichia coli/genética , Microbioma Gastrointestinal/genética , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/fisiología , Ratones , Transcriptoma
9.
Cell Rep ; 38(7): 110381, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35172154

RESUMEN

Cortical expansion in primate brains relies on enlargement of germinal zones during a prolonged developmental period. Although most mammals have two cortical germinal zones, the ventricular zone (VZ) and subventricular zone (SVZ), gyrencephalic species display an additional germinal zone, the outer subventricular zone (oSVZ), which increases the number and diversity of neurons generated during corticogenesis. How the oSVZ emerged during evolution is poorly understood, but recent studies suggest a role for non-coding RNAs, which allow tight genetic program regulation during development. Here, using in vivo functional genetics, single-cell RNA sequencing, live imaging, and electrophysiology to assess progenitor and neuronal properties in mice, we identify two oSVZ-expressed microRNAs (miRNAs), miR-137 and miR-122, which regulate key cellular features of cortical expansion. miR-137 promotes basal progenitor self-replication and superficial layer neuron fate, whereas miR-122 decreases the pace of neuronal differentiation. These findings support a cell-type-specific role of miRNA-mediated gene expression in cortical expansion.


Asunto(s)
Diferenciación Celular/genética , MicroARNs/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , ARN no Traducido/metabolismo , Animales , Proliferación Celular/genética , Reprogramación Celular/genética , Hurones , Células HEK293 , Humanos , Ventrículos Laterales , Ratones , MicroARNs/genética , Mitosis/genética , Neurogénesis/genética , Neuronas/metabolismo , ARN no Traducido/genética
10.
Cancer Res ; 82(4): 681-694, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-34916221

RESUMEN

Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches. SIGNIFICANCE: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.


Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Células Neoplásicas Circulantes/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , RNA-Seq/métodos , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Quinasa Tipo Polo 1
11.
Methods Mol Biol ; 2312: 171-192, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34228291

RESUMEN

Genome engineering technologies based on CRISPR-Cas systems are fueling efforts to study genotype-phenotype relationships in a high-throughput and multiplexed fashion. While many genome engineering technologies exist and provide a means to efficiently manipulate one or a few genes in a singular context-knockout, inhibition, or activation in a constitutive, conditional, or inducible manner-progress towards engineering complex cellular programs has been hampered by the lack of technologies that can integrate these functions within a unified framework. To address this challenge, our lab created single transcript CRISPR-Cas12a (SiT-Cas12a), which enables conditional, inducible, orthogonal, and massively multiplexed genome engineering of dozens, to potentially hundreds, of genomic targets in eukaryotic cells simultaneously-providing a novel way to interrogate and engineer complex genetic programs. In this chapter, we outline the utility of SiT-Cas12a in human cells and describe experimental procedures for executing massively multiplexed genome engineering experiments-including strategies for designing and assembling customized multiplexed CRISPR guide RNA arrays as well as validating and analyzing CRISPR guide RNA array processing and genome engineering outcomes.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endodesoxirribonucleasas/genética , Edición Génica , Regulación de la Expresión Génica , Transcripción Genética , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Técnicas de Cultivo de Célula , Endodesoxirribonucleasas/metabolismo , Células HEK293 , Humanos , Reacción en Cadena de la Polimerasa , Transfección
13.
iScience ; 24(1): 101935, 2021 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-33409479

RESUMEN

Genetic variation of the 16p11.2 deletion locus containing the KCTD13 gene and of CUL3 is linked with autism. This genetic connection suggested that substrates of a CUL3-KCTD13 ubiquitin ligase may be involved in disease pathogenesis. Comparison of Kctd13 mutant (Kctd13 -/- ) and wild-type neuronal ubiquitylomes identified adenylosuccinate synthetase (ADSS), an enzyme that catalyzes the first step in adenosine monophosphate (AMP) synthesis, as a KCTD13 ligase substrate. In Kctd13 -/- neurons, there were increased levels of succinyl-adenosine (S-Ado), a metabolite downstream of ADSS. Notably, S-Ado levels are elevated in adenylosuccinate lyase deficiency, a metabolic disorder with autism and epilepsy phenotypes. The increased S-Ado levels in Kctd13 -/- neurons were decreased by treatment with an ADSS inhibitor. Lastly, functional analysis of human KCTD13 variants suggests that KCTD13 variation may alter ubiquitination of ADSS. These data suggest that succinyl-AMP metabolites accumulate in Kctd13 -/- neurons, and this observation may have implications for our understanding of 16p11.2 deletion syndrome.

14.
Curr Opin Microbiol ; 59: 24-33, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32828048

RESUMEN

Advances in synthetic biology and microbiology have enabled the creation of engineered bacteria which can sense and report on intracellular and extracellular signals. When deployed in vivo these whole-cell bacterial biosensors can act as sentinels to monitor biomolecules of interest in human health and disease settings. This is particularly interesting in the context of the gut microbiota, which interacts extensively with the human host throughout time and transit of the gut and can be accessed from feces without requiring invasive collection. Leveraging rational engineering approaches for genetic circuits as well as an expanding catalog of disease-associated biomarkers, bacterial biosensors can act as non-invasive and easy-to-monitor reporters of the gut. Here, we summarize recent engineering approaches applied in vivo in animal models and then highlight promising technologies for designing the next generation of bacterial biosensors.


Asunto(s)
Bacterias , Técnicas Biosensibles , Microbioma Gastrointestinal , Tracto Gastrointestinal , Organismos Modificados Genéticamente , Animales , Bacterias/genética , Bacterias/metabolismo , Técnicas Biosensibles/métodos , Heces/microbiología , Microbioma Gastrointestinal/genética , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Humanos
15.
Nat Protoc ; 15(2): 513-539, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31925399

RESUMEN

It is difficult to elucidate the transcriptional history of a cell using current experimental approaches, as they are destructive in nature and therefore describe only a moment in time. To overcome these limitations, we recently established Record-seq, a technology that enables transcriptional recording by CRISPR spacer acquisition from RNA. The recorded transcriptomes are recovered by SENECA, a method that selectively amplifies expanded CRISPR arrays, followed by deep sequencing. The resulting CRISPR spacers are aligned to the host genome, thereby enabling transcript quantification and associated analyses. Here, we describe the experimental procedures of the Record-seq workflow as well as subsequent data analysis. Beginning with the experimental design, Record-seq data can be obtained and analyzed within 1-2 weeks.


Asunto(s)
Análisis de Secuencia de ADN/métodos , Transcripción Genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Escherichia coli/genética
17.
Nat Methods ; 16(9): 887-893, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31406383

RESUMEN

The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.


Asunto(s)
Sistemas CRISPR-Cas , Endonucleasas/metabolismo , Edición Génica , Redes Reguladoras de Genes , Ingeniería Genética , Genoma Humano , ARN Guía de Kinetoplastida/genética , Acidaminococcus/enzimología , Endonucleasas/genética , Células HEK293 , Humanos , Plásmidos/genética , Activación Transcripcional
18.
Nat Protoc ; 14(7): 2259, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-30349047

RESUMEN

In the published version of this paper, Step 64 of the Procedure reads, "Refer to Steps 37-39 for NGS analysis of the sgRNA distribution." This step should refer the reader to Steps 35-39. This text has not been corrected in the original paper.

19.
Nature ; 562(7727): 380-385, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30283135

RESUMEN

The ability to record transcriptional events within a cell over time would help to elucidate how molecular events give rise to complex cellular behaviours and states. However, current molecular recording technologies capture only a small set of defined stimuli. Here we use CRISPR spacer acquisition to capture and convert intracellular RNAs into DNA, enabling DNA-based storage of transcriptional information. In Escherichia coli, we show that defined stimuli, such as an RNA virus or arbitrary sequences, as well as complex stimuli, such as oxidative stress, result in quantifiable transcriptional records that are stored within a population of cells. We demonstrate that the transcriptional records enable us to classify and describe complex cellular behaviours and to identify the precise genes that orchestrate differential cellular responses. In the future, CRISPR spacer acquisition-mediated recording of RNA followed by deep sequencing (Record-seq) could be used to reconstruct transcriptional histories that describe complex cell behaviours or pathological states.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , ARN/análisis , ARN/genética , Transcripción Genética/genética , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Clostridiales/enzimología , Clostridiales/genética , ADN/análisis , ADN/genética , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Genes Bacterianos/genética , Herbicidas/farmacología , Estrés Oxidativo/genética , Paraquat/farmacología
20.
Sci Adv ; 4(2): eaao5508, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29503867

RESUMEN

Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas some mutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct high-throughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adeno-associated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice. All mice that received the AAV-mTSG library developed liver cancer and died within 4 months. We used molecular inversion probe sequencing of the sgRNA target sites to chart the mutational landscape of these tumors, revealing the functional consequence of multiple variants in driving liver tumorigenesis in immunocompetent mice. AAV-mediated autochthonous CRISPR screens provide a powerful means for mapping a provisional functional cancer genome atlas of tumor suppressors in vivo.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Dependovirus/genética , Pruebas Genéticas , Genoma , Hígado/patología , Neoplasias/genética , Animales , Carcinogénesis/patología , Genes Supresores de Tumor , Ratones , Mutación/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA