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1.
Gut ; 69(2): 231-242, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31068366

RESUMO

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer mortality. Previous studies have shown that hepatocyte nuclear factor-4α (HNF4α) is specifically overexpressed in GC and functionally required for GC development. In this study, we investigated, on a genome-wide scale, target genes of HNF4α and oncogenic pathways driven by HNF4α and HNF4α target genes. DESIGN: We performed HNF4α chromatin immunoprecipitation followed by sequencing across multiple GC cell lines, integrating HNF4α occupancy data with (epi)genomic and transcriptome data of primary GCs to define HNF4α target genes of in vitro and in vivo relevance. To investigate mechanistic roles of HNF4α and HNF4α targets, we performed cancer metabolic measurements, drug treatments and functional assays including murine xenograft experiments. RESULTS: Gene expression analysis across 19 tumour types revealed HNF4α to be specifically upregulated in GCs. Unbiased pathway analysis revealed organic acid metabolism as the top HNF4α-regulated pathway, orthogonally supported by metabolomic analysis. Isocitrate dehydrogenase 1 (IDH1) emerged as a convergent HNF4α direct target gene regulating GC metabolism. We show that wild-type IDH1 is essential for GC cell survival, and that certain GC cells can be targeted by IDH1 inhibitors. CONCLUSIONS: Our results highlight a role for HNF4α in sustaining GC oncogenic metabolism, through the regulation of IDH1. Drugs targeting wild-type IDH1 may thus have clinical utility in GCs exhibiting HNF4α overexpression, expanding the role of IDH1 in cancer beyond IDH1/2 mutated malignancies.


Assuntos
Fator 4 Nuclear de Hepatócito/genética , Isocitrato Desidrogenase/metabolismo , Neoplasias Gástricas/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Marcação de Genes/métodos , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Camundongos Endogâmicos NOD , Terapia de Alvo Molecular/métodos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Life Sci ; 239: 117008, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31669240

RESUMO

OBJECTIVE: We aimed to explore the expression level and biological function of miR-145-5p in preeclampsia (PE). METHODS: The differentially expressed miRNA/mRNA between normal placentas and PE placentas were screened using the GSE84260 and GSE73374 datasets from the Gene Expression Omnibus Database. The expression of miR-145-5p in PE placentas was detected by qRT-PCR. The CCK-8 assay, wound healing and transwell were carried out to determine the cell growth, migration and invasion when miR-145-5p was overexpressed or inhibited. The real-time quantitative PCR (qRT-PCR), Western Blot and dual-luciferase reporter assays were conducted to preliminarily explore possible mechanisms. RESULTS: A total of 33 miRNAs were found significantly differentially expressed in PE patients, 19 were significantly upregulated and 14 were significantly downregulated. The relative miR-145-5p expression was lower in PE placentas than normal placentas. The viability and invasion were suppressed when miR-145-5p was inhibited in trophoblasts cells, while miR-145-5p overexpression promoted the effectiveness. In addition, mRNA and protein expression of FLT1 in HTR-8/SVneo cell was also downregulated with miR-145-5p overexpression, suggesting that FLT1 is the target gene of miR-145-5p. Consistent with miR-145-5p overexpression, the mRNA and protein expression of FLT1 also were upregulated with miR-145-5p interference. Furthermore, the expression of miR-145-5p was regulated by the Hypoxic conditions. CONCLUSIONS: In conclusion, the results showed miR-145-5p may participate in PE development by affecting the proliferation and invasion of trophoblast cells. This is a new perspective to understand the etiology and pathogenesis of PE, which may provide a new breakthrough for the early prediction and diagnosis of PE.


Assuntos
MicroRNAs/genética , Trofoblastos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Adulto , Divisão Celular , Hipóxia Celular , Movimento Celular , Células Cultivadas , Bases de Dados Genéticas , Feminino , Redes Reguladoras de Genes , Marcação de Genes , Humanos , Placenta/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Gravidez
3.
Nat Commun ; 10(1): 4439, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570731

RESUMO

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA , Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos , Integração Viral/genética , Animais , Bacteriófago lambda/genética , Encéfalo , Linhagem Celular , Mapeamento Cromossômico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cóclea , Endonucleases , Marcação de Genes/métodos , Terapia Genética/métodos , Genoma , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculos , Neurônios/virologia , Reparo Gênico Alvo-Dirigido/métodos , Resultado do Tratamento
5.
Immunity ; 51(4): 593-594, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618647
6.
Nat Biotechnol ; 37(9): 1041-1048, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477922

RESUMO

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1-4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Animais , Clonagem Molecular , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Células HEK293 , Humanos , Petromyzon , Conformação Proteica , RNA , RNA Guia/genética , Streptococcus pyogenes , Transcriptoma
7.
Life Sci Alliance ; 2(4)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31409625

RESUMO

Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division in male gametocyte together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated, but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy, and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not co-localise with kinetochores in the nucleus but instead revealed a dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We, thus, uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.


Assuntos
Corpos Basais/metabolismo , Flagelos/fisiologia , Cinesina/metabolismo , Malária/transmissão , Plasmodium malariae/fisiologia , Animais , Axonema/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Cinesina/genética , Cinetocoros/metabolismo , Microscopia Eletrônica , Mitose
8.
Genes Dev ; 33(17-18): 1265-1279, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31395741

RESUMO

Chromosomal rearrangements of the mixed lineage leukemia (MLL) gene occur in ∼10% of B-cell acute lymphoblastic leukemia (B-ALL) and define a group of patients with dismal outcomes. Immunohistochemical staining of bone marrow biopsies from most of these patients revealed aberrant expression of BCL6, a transcription factor that promotes oncogenic B-cell transformation and drug resistance in B-ALL. Our genetic and ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analyses showed that MLL-AF4 and MLL-ENL fusions directly bound to the BCL6 promoter and up-regulated BCL6 expression. While oncogenic MLL fusions strongly induced aberrant BCL6 expression in B-ALL cells, germline MLL was required to up-regulate Bcl6 in response to physiological stimuli during normal B-cell development. Inducible expression of Bcl6 increased MLL mRNA levels, which was reversed by genetic deletion and pharmacological inhibition of Bcl6, suggesting a positive feedback loop between MLL and BCL6. Highlighting the central role of BCL6 in MLL-rearranged B-ALL, conditional deletion and pharmacological inhibition of BCL6 compromised leukemogenesis in transplant recipient mice and restored sensitivity to vincristine chemotherapy in MLL-rearranged B-ALL patient samples. Oncogenic MLL fusions strongly induced transcriptional activation of the proapoptotic BH3-only molecule BIM, while BCL6 was required to curb MLL-induced expression of BIM. Notably, peptide (RI-BPI) and small molecule (FX1) BCL6 inhibitors derepressed BIM and synergized with the BH3-mimetic ABT-199 in eradicating MLL-rearranged B-ALL cells. These findings uncover MLL-dependent transcriptional activation of BCL6 as a previously unrecognized requirement of malignant transformation by oncogenic MLL fusions and identified BCL6 as a novel target for the treatment of MLL-rearranged B-ALL.


Assuntos
Regulação Leucêmica da Expressão Gênica , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Animais , Biomarcadores Tumorais/genética , Sobrevivência Celular/genética , Células Cultivadas , Deleção de Genes , Marcação de Genes , Humanos , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Prognóstico , Regiões Promotoras Genéticas/genética
10.
Sheng Li Xue Bao ; 71(4): 588-596, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440756

RESUMO

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2-/Y mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2-/Y mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Camundongos Knockout , RNA Guia/genética , Animais , Feminino , Marcação de Genes , Masculino , Camundongos
11.
Cell Mol Life Sci ; 76(24): 4861-4867, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31367845

RESUMO

RNAs are responsible for mediating genetic information flow within the cell. RNA splicing, modification, trafficking, translation, and stability are all controlled at the transcript level. However, biological tools to study and manipulate them in a programmable fashion are currently limited. In this review, we summarize recent advances regarding available RNA-targeting systems discovered so far, including CRISPR-based technologies-Cas9 and Cas13, and programmable RNA-binding proteins-PUF and PPR. These tools allow transcript-specific manipulation in gene expression.


Assuntos
RNA Guia/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , Marcação de Genes/tendências , Humanos
12.
Cell Mol Life Sci ; 76(24): 4869-4886, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31377844

RESUMO

The chemokine system mediates acute inflammation by driving leukocyte migration to damaged or infected tissues. However, elevated expression of chemokines and their receptors can contribute to chronic inflammation and malignancy. Thus, great effort has been taken to target these molecules. The first hint of the druggability of the chemokine system was derived from the role of chemokine receptors in HIV infection. CCR5 and CXCR4 function as essential co-receptors for HIV entry, with the former accounting for most new HIV infections worldwide. Not by chance, an anti-CCR5 compound, maraviroc, was the first FDA-approved chemokine receptor-targeting drug. CCR5, by directing leukocytes to sites of inflammation and regulating their activation, also represents an important player in the inflammatory response. This function is shared with CCR2 and its selective ligand CCL2, which constitute the primary chemokine axis driving the recruitment of monocytes/macrophages to inflammatory sites. Both receptors are indeed involved in the pathogenesis of several immune-mediated diseases, and dual CCR5/CCR2 targeting is emerging as a more efficacious strategy than targeting either receptor alone in the treatment of complex human disorders. In this review, we focus on the distinctive and complementary contributions of CCR5 and CCR2/CCL2 in HIV infection, multiple sclerosis, liver fibrosis and associated hepatocellular carcinoma. The emerging therapeutic approaches based on the inhibition of these chemokine axes are highlighted.


Assuntos
Quimiocina CCL2/genética , Inflamação/genética , Receptores CCR2/genética , Receptores CCR5/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Marcação de Genes , HIV/genética , HIV/patogenicidade , Infecções por HIV/genética , Infecções por HIV/terapia , Infecções por HIV/virologia , Humanos , Inflamação/terapia , Cirrose Hepática/genética , Cirrose Hepática/terapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Esclerose Múltipla/genética , Esclerose Múltipla/terapia
13.
Nat Biotechnol ; 37(9): 1070-1079, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31332326

RESUMO

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.


Assuntos
Adenosina Desaminase/metabolismo , Evolução Molecular Direcionada , Edição de Genes , Adenosina Desaminase/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Humanos , Mutação INDEL , Camundongos
14.
Nat Neurosci ; 22(8): 1345-1356, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285614

RESUMO

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.


Assuntos
Dependovirus/genética , Marcação de Genes/métodos , Neuroglia/virologia , Neurônios/virologia , Animais , Técnicas de Transferência de Genes , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Retina/virologia
15.
Appl Microbiol Biotechnol ; 103(17): 6919-6932, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31332488

RESUMO

Filamentous fungi play an important role in human health and industrial/agricultural production. With the increasing number of full genomes available for fungal species, the study of filamentous fungi has brought about a wider range of genetic manipulation opportunities. However, the utilization of traditional methods to study fungi is time consuming and laborious. Recent rapid progress and wide application of a versatile genome editing technology, i.e., the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-related nuclease 9) system, has revolutionized biological research and has many innovative applications in a wide range of fields showing great promise in research and application of filamentous fungi. In this review, we introduce the CRISPR/Cas9 genome editing technology focusing on its application in research of filamentous fungi and we discuss the general considerations of genome editing using CRISPR/Cas9 system illustrating vector construction, multiple editing strategies, technical consideration of different sizes of homology arms on genome editing efficiency, off-target effects, and different transformation methodologies. In addition, we discuss the challenges encountered using CRISPR/Cas9 technology and give the perspectives of future applications of CRISPR/Cas9 technology for basic research and practical application of filamentous fungi.


Assuntos
Sistemas CRISPR-Cas , Fungos/genética , Edição de Genes , Genoma Fúngico/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Marcação de Genes , Microbiologia Industrial , Mutação , RNA Guia/genética , Transformação Genética
16.
PLoS Biol ; 17(7): e3000350, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31265461

RESUMO

Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in one step and screened for bone-development-related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a, which are required for bone development, and Rln1 and Irx5, which had not been previously considered. Whereas Rln1-/- mice exhibited small skeletal size only at birth, Irx5-/- mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice.


Assuntos
Desenvolvimento Ósseo/genética , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes/métodos , Testes Genéticos/métodos , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Haploidia , Fatores de Crescimento de Células Hematopoéticas/genética , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Knockout , Relaxina/genética , Relaxina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Int J Mol Sci ; 20(15)2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31357652

RESUMO

CRISPR/Cas technology holds promise for the development of therapies to treat inherited diseases. Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder with a variable multisystemic character for which no cure is yet available. Here, we review CRISPR/Cas-mediated approaches that target the unstable (CTG•CAG)n repeat in the DMPK/DM1-AS gene pair, the autosomal dominant mutation that causes DM1. Expansion of the repeat results in a complex constellation of toxicity at the DNA level, an altered transcriptome and a disturbed proteome. To restore cellular homeostasis and ameliorate DM1 disease symptoms, CRISPR/Cas approaches were directed at the causative mutation in the DNA and the RNA. Specifically, the triplet repeat has been excised from the genome by several laboratories via dual CRISPR/Cas9 cleavage, while one group prevented transcription of the (CTG)n repeat through homology-directed insertion of a polyadenylation signal in DMPK. Independently, catalytically deficient Cas9 (dCas9) was recruited to the (CTG)n repeat to block progression of RNA polymerase II and a dCas9-RNase fusion was shown to degrade expanded (CUG)n RNA. We compare these promising developments in DM1 with those in other microsatellite instability diseases. Finally, we look at hurdles that must be taken to make CRISPR/Cas-mediated editing a therapeutic reality in patients.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Terapia Genética , Distrofia Miotônica/genética , Distrofia Miotônica/terapia , Animais , Terapia Baseada em Transplante de Células e Tecidos , Marcação de Genes , Estudos de Associação Genética , Loci Gênicos , Predisposição Genética para Doença , Humanos , Expansão das Repetições de Trinucleotídeos , Repetições de Trinucleotídeos
18.
Methods Mol Biol ; 1982: 623-665, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172498

RESUMO

Chronic granulomatous disease (CGD) is an immune deficiency characterized by defects in the production of microbicidal reactive oxygen species (ROS) by the phagocytic oxidase (phox) enzyme complex in neutrophils. We have previously described targeted gene editing strategies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases for gene targeting with homology-directed repair in CGD patient stem cells to achieve functional restoration of expression of phox genes and NADPH oxidase activity in differentiated neutrophils. In this chapter, we describe detailed protocols for targeted gene editing in human-induced pluripotent stem cells and hematopoietic stem cells and for subsequent differentiation of these stem cells into mature neutrophils, as well as assays to characterize neutrophil identity and function including flow cytometry analysis of neutrophil surface markers, intracellular staining for phox proteins, and analysis of ROS generation.


Assuntos
Edição de Genes , Doença Granulomatosa Crônica/genética , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Edição de Genes/métodos , Ordem dos Genes , Marcação de Genes , Vetores Genéticos , Doença Granulomatosa Crônica/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , RNA Guia , Espécies Reativas de Oxigênio/metabolismo
19.
Life Sci Alliance ; 2(3)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31196871

RESUMO

CRISPR-Cas9-based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms-often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.


Assuntos
Cromossomos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Drosophila/genética , Edição de Genes , Expressão Gênica , Marcação de Genes , Genes Reporter , Conformação de Ácido Nucleico , Fenótipo , RNA Guia/genética
20.
PLoS Genet ; 15(6): e1007721, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199803

RESUMO

B-cell activation yields abundant cell death in parallel to clonal amplification and remodeling of immunoglobulin (Ig) genes by activation-induced deaminase (AID). AID promotes affinity maturation of Ig variable regions and class switch recombination (CSR) in mature B lymphocytes. In the IgH locus, these processes are under control of the 3' regulatory region (3'RR) super-enhancer, a region demonstrated in the mouse to be both transcribed and itself targeted by AID-mediated recombination. Alternatively to CSR, IgH deletions joining Sµ to "like-switch" DNA repeats that flank the 3' super-enhancer can thus accomplish so-called "locus suicide recombination" (LSR) in mouse B-cells. Using an optimized LSR-seq high throughput method, we now show that AID-mediated LSR is evolutionarily conserved and also actively occurs in humans, providing an activation-induced cell death pathway in multiple conditions of B-cell activation. LSR either focuses on the functional IgH allele or is bi-allelic, and its signature is mainly detected when LSR is ongoing while it vanishes from fully differentiated plasma cells or from "resting" blood memory B-cells. Highly diversified breakpoints are distributed either within the upstream (3'RR1) or downstream (3'RR2) copies of the IgH 3' super-enhancer and all conditions activating CSR in vitro also seem to trigger LSR although TLR ligation appeared the most efficient. Molecular analysis of breakpoints and junctions confirms that LSR is AID-dependent and reveals junctional sequences somehow similar to CSR junctions but with increased usage of microhomologies.


Assuntos
Linfócitos B/imunologia , Citidina Desaminase/genética , Região de Troca de Imunoglobulinas/genética , Imunoglobulinas/imunologia , Alelos , Animais , Diferenciação Celular/genética , Citidina Desaminase/imunologia , Marcação de Genes , Humanos , Região de Troca de Imunoglobulinas/imunologia , Tecido Linfoide/imunologia , Camundongos , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Sequências Reguladoras de Ácido Nucleico
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