Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 104
Filtrar
1.
Immunity ; 57(5): 1005-1018.e7, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38697116

RESUMO

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.


Assuntos
Fator de Ligação a CCCTC , Diferenciação Celular , Interferon gama , Interleucina 22 , Interleucinas , Células Th1 , Animais , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Células Th1/imunologia , Camundongos , Diferenciação Celular/imunologia , Interferon gama/metabolismo , Sítios de Ligação , Interleucinas/metabolismo , Interleucinas/genética , Elementos Facilitadores Genéticos/genética , Camundongos Endogâmicos C57BL , Cromatina/metabolismo , Toxoplasmose/imunologia , Toxoplasmose/parasitologia , Toxoplasmose/genética , Regulação da Expressão Gênica , Toxoplasma/imunologia , Citocinas/metabolismo , Linhagem da Célula , Células Th17/imunologia
2.
Nat Immunol ; 25(4): 644-658, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38503922

RESUMO

The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially localized multicellular 'immunity hubs' defined by expression of the T cell-attracting chemokines CXCL10/CXCL11 and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens and found an association with beneficial response to PD-1 blockade. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcome. This hub is distinct from mature tertiary lymphoid structures and is enriched for stem-like TCF7+PD-1+CD8+ T cells, activated CCR7+LAMP3+ dendritic cells and CCL19+ fibroblasts as well as chemokines that organize these cells. Within the stem-immunity hub, we find preferential interactions between CXCL10+ macrophages and TCF7-CD8+ T cells as well as between mature regulatory dendritic cells and TCF7+CD4+ and regulatory T cells. These results provide a picture of the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes.


Assuntos
Neoplasias Pulmonares , Humanos , Linfócitos T CD8-Positivos , Receptor de Morte Celular Programada 1 , Quimiocinas/metabolismo , Imunoterapia/métodos , Microambiente Tumoral
3.
bioRxiv ; 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38105940

RESUMO

Purpose: Dysregulation of viral-like repeat RNAs are a common feature across many malignancies that are linked with immunological response, but the characterization of these in hepatocellular carcinoma (HCC) is understudied. In this study, we performed RNA in situ hybridization (RNA-ISH) of different repeat RNAs, immunohistochemistry (IHC) for immune cell subpopulations, and spatial transcriptomics to understand the relationship of HCC repeat expression, immune response, and clinical outcomes. Experimental Design: RNA-ISH for LINE1, HERV-K, HERV-H, and HSATII repeats and IHC for T-cell, Treg, B-cell, macrophage, and immune checkpoint markers were performed on 43 resected HCC specimens. Spatial transcriptomics on tumor and vessel regions of interest was performed on 28 specimens from the same cohort. Results: High HERV-K and high LINE1 expression were both associated with worse overall survival. There was a positive correlation between LINE1 expression and FOXP3 T-regulatory cells (r = 0.51 p < 0.001) as well as expression of the TIM3 immune checkpoint (r = 0.34, p = 0.03). Spatial transcriptomic profiling of HERV-K high and LINE-1 high tumors identified elevated expression of multiple genes previously associated with epithelial mesenchymal transition, cellular proliferation, and worse overall prognosis in HCC including SSX1, MAGEC2, and SPINK1. Conclusion: Repeat RNAs may serve as useful prognostic biomarkers in HCC and may also serve as novel therapeutic targets. Additional study is needed to understand the mechanisms by which repeat RNAs impact HCC tumorigenesis.

4.
bioRxiv ; 2023 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-37961446

RESUMO

DNA looping is vital for establishing many enhancer-promoter interactions. While CTCF is known to anchor many cohesin-mediated loops, the looped chromatin fiber appears to predominantly exist in a poorly characterized actively extruding state. To better characterize extruding chromatin loop structures, we used CTCF MNase HiChIP data to determine both CTCF binding at high resolution and 3D contact information. Here we present FactorFinder, a tool that identifies CTCF binding sites at near base-pair resolution. We leverage this substantial advance in resolution to determine that the fully extruded (CTCF-CTCF) state is rare genome-wide with locus-specific variation from ~1-10%. We further investigate the impact of chromatin state on loop extrusion dynamics, and find that active enhancers and RNA Pol II impede cohesin extrusion, facilitating an enrichment of enhancer-promoter contacts in the partially extruded loop state. We propose a model of topological regulation whereby the transient, partially extruded states play active roles in transcription.

5.
bioRxiv ; 2023 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-37786671

RESUMO

Enhancers possess both structural elements mediating promoter looping and functional elements mediating gene expression. Traditional models of enhancer-mediated gene regulation imply genomic overlap or immediate adjacency of these elements. We test this model by combining densely-tiled CRISPRa screening with nucleosome-resolution Region Capture Micro-C topology analysis. Using this integrated approach, we comprehensively define the cis-regulatory landscape for the tumor suppressor PTEN, identifying and validating 10 distinct enhancers and defining their 3D spatial organization. Unexpectedly, we identify several long-range functional enhancers whose promoter proximity is facilitated by chromatin loop anchors several kilobases away, and demonstrate that accounting for this spatial separation improves the computational prediction of validated enhancers. Thus, we propose a new model of enhancer organization incorporating spatial separation of essential functional and structural components.

6.
Blood ; 142(21): 1831-1844, 2023 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-37699201

RESUMO

Severe acute graft-versus-host disease (aGVHD) is associated with significant mortality and morbidity, especially in steroid-resistant (SR) cases. Spatial transcriptomic technology can elucidate tissue-based interactions in vivo and possibly identify predictors of treatment response. Tissue sections from 32 treatment-naïve patients with biopsy-confirmed lower gastrointestinal (GI) aGVHD were obtained. The GeoMx digital spatial profiler was used to capture transcriptome profiles of >18 000 genes from different foci of immune infiltrates, colonic epithelium, and vascular endothelium. Each tissue compartment sampled showed 2 distinct clusters that were analyzed for differential expression and spatially resolved correlation of gene signatures. Classic cell-mediated immunity signatures, normal differentiated epithelial cells, and inflamed vasculature dominated foci sampled from steroid-sensitive cases. In contrast, a neutrophil predominant noncanonical inflammation with regenerative epithelial cells and some indication of angiogenic endothelial response was overrepresented in areas from SR cases. Evaluation of potential prognostic biomarkers identified ubiquitin specific peptidase 17-like (USP17L) family of genes as being differentially expressed in immune cells from patients with worsened survival. In summary, we demonstrate distinct tissue niches with unique gene expression signatures within lower GI tissue from patients with aGVHD and provide evidence of a potential prognostic biomarker.


Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Transcriptoma , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/genética , Imunidade Celular , Esteroides/uso terapêutico , Mucosa Intestinal , Doença Aguda
8.
Cell ; 186(13): 2765-2782.e28, 2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37327786

RESUMO

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.


Assuntos
Metilação de DNA , Neoplasias da Próstata , Animais , Humanos , Masculino , Camundongos , Carcinogênese/genética , DNA , Epigênese Genética , Neoplasias da Próstata/genética , Células Neoplásicas Circulantes
9.
bioRxiv ; 2023 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-37066412

RESUMO

The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially-localized multicellular 'immunity hubs' defined by expression of the T cell-attracting chemokines CXCL10/CXCL11 and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens, and found that they were associated with beneficial responses to PD-1-blockade. Immunity hubs were enriched for many interferon-stimulated genes, T cells in multiple differentiation states, and CXCL9/10/11 + macrophages that preferentially interact with CD8 T cells. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcomes, distinct from mature tertiary lymphoid structures, and enriched for stem-like TCF7+PD-1+ CD8 T cells and activated CCR7 + LAMP3 + dendritic cells, as well as chemokines that organize these cells. These results elucidate the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes.

10.
Sci Adv ; 9(13): eabo3789, 2023 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-37000878

RESUMO

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.


Assuntos
Sarcoma de Ewing , Criança , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Cromatina/genética , Linhagem Celular Tumoral , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sítios de Ligação , Diferenciação Celular , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regulação Neoplásica da Expressão Gênica
11.
Science ; 378(6626): 1276-1277, 2022 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-36548410

RESUMO

Global methylation changes in aging cells affect cancer risk and tissue homeostasis.


Assuntos
Envelhecimento , Metilação de DNA , Epigênese Genética , Mitose , Neoplasias , Humanos , Envelhecimento/genética , Senescência Celular , Epigenômica , Neoplasias/genética
12.
Nat Commun ; 13(1): 4199, 2022 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-35859152

RESUMO

The genome can be divided into two spatially segregated compartments, A and B, which partition active and inactive chromatin states. While constitutive heterochromatin is predominantly located within the B compartment near the nuclear lamina, facultative heterochromatin marked by H3K27me3 spans both compartments. How epigenetic modifications, compartmentalization, and lamina association collectively maintain heterochromatin architecture remains unclear. Here we develop Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) to jointly measure chromosome conformation, DNA methylation, and lamina positioning. Through LIMe-Hi-C, we identify topologically distinct sub-compartments with high levels of H3K27me3 and differing degrees of lamina association. Inhibition of Polycomb repressive complex 2 (PRC2) reveals that H3K27me3 is essential for sub-compartment segregation. Unexpectedly, PRC2 inhibition promotes lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with this repositioning, genes originally marked with H3K27me3 in the B compartment, but not the A compartment, remain largely repressed, suggesting that constitutive heterochromatin spreading can compensate for H3K27me3 loss at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with lamina association are fundamental features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how compartmentalization and lamina association represent distinct but interdependent modes of heterochromatin regulation.


Assuntos
Heterocromatina , Histonas , Núcleo Celular/metabolismo , Metilação de DNA , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo
14.
Mol Ther Nucleic Acids ; 28: 450-461, 2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35505961

RESUMO

Presenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-ß (Aß) peptide that deposits as plaques in the Alzheimer's disease (AD) brain. The M146L mutation in the PS1 gene (PSEN1) leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aß42. This change is evident not only in the brain but also in peripheral cells of mutation carriers. In this study we used the CRISPR-Cas9 system from Streptococcus pyogenes to selectively disrupt the PSEN1 M146L allele in human fibroblasts. A disruption of more than 50% of mutant alleles was observed in all CRISPR-Cas9-treated samples, resulting in reduced extracellular Aß42/40 ratios. Fluorescence resonance energy transfer-based conformation and western blot analyses indicated that CRISPR-Cas9 treatment also affects the overall PS1 conformation and reduces PS1 levels. Moreover, our guide RNA did not lead to any detectable editing at the highest-ranking candidate off-target sites identified by ONE-seq and CIRCLE-seq. Overall, our data support the effectiveness of CRISPR-Cas9 in selectively targeting the PSEN1 M146L allele and counteracting the AD-associated phenotype. We believe that this system could be developed into a therapeutic strategy for patients with this and other dominant mutations leading to early-onset AD.

15.
Nat Commun ; 13(1): 2267, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35477713

RESUMO

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.


Assuntos
Sarcoma de Células Claras , Neoplasias de Tecidos Moles , Carcinogênese/genética , Cromatina/genética , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes , Proteína EWS de Ligação a RNA/genética , Sarcoma de Células Claras/genética , Sarcoma de Células Claras/patologia , Neoplasias de Tecidos Moles/genética
16.
Commun Biol ; 5(1): 213, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35260776

RESUMO

Aberrant methylation of genomic DNA has been reported in many cancers. Specific DNA methylation patterns have been shown to provide clinically useful prognostic information and define molecular disease subtypes with different response to therapy and long-term outcome. Osteosarcoma is an aggressive malignancy for which approximately half of tumors recur following standard combined surgical resection and chemotherapy. No accepted prognostic factor save tumor necrosis in response to adjuvant therapy currently exists, and traditional genomic studies have thus far failed to identify meaningful clinical associations. We studied the genome-wide methylation state of primary tumors and tested how they predict patient outcomes. We discovered relative genomic hypomethylation to be strongly predictive of response to standard chemotherapy. Recurrence and survival were also associated with genomic methylation, but through more site-specific patterns. Furthermore, the methylation patterns were reproducible in three small independent clinical datasets. Downstream transcriptional, in vitro, and pharmacogenomic analysis provides insight into the clinical translation of the methylation patterns. Our findings suggest the assessment of genomic methylation may represent a strategy for stratifying patients for the application of alternative therapies.


Assuntos
Neoplasias Ósseas , Osteossarcoma , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , DNA , Metilação de DNA , Humanos , Osteossarcoma/genética , Osteossarcoma/patologia , Prognóstico
17.
Cancer Discov ; 12(6): 1462-1481, 2022 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-35320348

RESUMO

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements. SIGNIFICANCE: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.


Assuntos
Neoplasias Colorretais , DNA Polimerase Dirigida por RNA , Animais , Antivirais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , DNA , Humanos , Interferons/metabolismo , Lamivudina , Estágios do Ciclo de Vida , RNA , DNA Polimerase Dirigida por RNA/metabolismo , Proteína Supressora de Tumor p53/genética
18.
Blood ; 139(16): 2534-2546, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-35030251

RESUMO

Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.


Assuntos
Anemia , Fator de Transcrição GATA1 , Diferenciação Celular/genética , Cromatina/genética , Imunoprecipitação da Cromatina , Eritropoese/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Humanos
19.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34903652

RESUMO

The current high mortality of human lung cancer stems largely from the lack of feasible, early disease detection tools. An effective test with serum metabolomics predictive models able to suggest patients harboring disease could expedite triage patient to specialized imaging assessment. Here, using a training-validation-testing-cohort design, we establish our high-resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS)-based metabolomics predictive models to indicate lung cancer presence and patient survival using serum samples collected prior to their disease diagnoses. Studied serum samples were collected from 79 patients before (within 5.0 y) and at lung cancer diagnosis. Disease predictive models were established by comparing serum metabolomic patterns between our training cohorts: patients with lung cancer at time of diagnosis, and matched healthy controls. These predictive models were then applied to evaluate serum samples of our validation and testing cohorts, all collected from patients before their lung cancer diagnosis. Our study found that the predictive model yielded values for prior-to-detection serum samples to be intermediate between values for patients at time of diagnosis and for healthy controls; these intermediate values significantly differed from both groups, with an F1 score = 0.628 for cancer prediction. Furthermore, values from metabolomics predictive model measured from prior-to-diagnosis sera could significantly predict 5-y survival for patients with localized disease.


Assuntos
Detecção Precoce de Câncer/métodos , Neoplasias Pulmonares/diagnóstico , Espectroscopia de Ressonância Magnética , Metabolômica , Idoso , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/metabolismo , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes
20.
Nat Protoc ; 16(12): 5592-5615, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34773119

RESUMO

Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Humano , Reação em Cadeia da Polimerase/métodos , RNA Guia de Cinetoplastídeos/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Primers do DNA/síntese química , Primers do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/química , Eletroporação/métodos , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , RNA Guia de Cinetoplastídeos/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA