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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.
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
3.
Mol Cell ; 76(5): 826-837.e11, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31607545

RESUMO

The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Marcação de Genes/métodos , Estabilidade de RNA , Vírus de RNA/enzimologia , RNA Viral/metabolismo , Células A549 , Animais , Proteínas Associadas a CRISPR/genética , Cães , Escherichia coli/enzimologia , Escherichia coli/genética , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Vírus de RNA/genética , RNA Viral/genética , Células Vero
4.
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
5.
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
6.
Curr Protoc Mol Biol ; 127(1): e89, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31237422

RESUMO

Genetic tools for specific perturbation of endogenous gene expression are highly desirable for interrogation of plant gene functions and improvement of crop traits. Synthetic transcriptional activators derived from the CRISPR/Cas9 system are emerging as powerful new tools for activating the endogenous expression of genes of interest in plants. These synthetic constructs, generated by tethering transcriptional activation domains to a nuclease-dead Cas9 (dCas9), can be directed to the promoters of endogenous target genes by single guide RNAs (sgRNAs) to activate transcription. Here, we provide a detailed protocol for targeted transcriptional activation in plants using a recently developed, highly potent dCas9 gene activator construct referred to as dCas9-TV. This protocol covers selection of sgRNA targets, construction of sgRNA expression cassettes, and screening for an optimal sgRNA using a protoplast-based promoter-luciferase assay. Finally, the dCas9-TV gene activator coupled with the optimal sgRNA is delivered into plants via Agrobacterium-mediated transformation, thereby enabling robust upregulation of target gene expression in transgenic Arabidopsis and rice plants. © 2019 by John Wiley & Sons, Inc.


Assuntos
Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Marcação de Genes/métodos , Genes Sintéticos/genética , Oryza/genética , Ativação Transcricional , Sistemas CRISPR-Cas/genética , Genes de Plantas/genética , Regiões Promotoras Genéticas , Protoplastos/metabolismo , RNA Guia/genética
7.
BMC Biotechnol ; 19(1): 40, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248401

RESUMO

BACKGROUND: Natural variations in a genome can drastically alter the CRISPR-Cas9 off-target landscape by creating or removing sites. Despite the resulting potential side-effects from such unaccounted for sites, current off-target detection pipelines are not equipped to include variant information. To address this, we developed VARiant-aware detection and SCoring of Off-Targets (VARSCOT). RESULTS: VARSCOT identifies only 0.6% of off-targets to be common between 4 individual genomes and the reference, with an average of 82% of off-targets unique to an individual. VARSCOT is the most sensitive detection method for off-targets, finding 40 to 70% more experimentally verified off-targets compared to other popular software tools and its machine learning model allows for CRISPR-Cas9 concentration aware off-target activity scoring. CONCLUSIONS: VARSCOT allows researchers to take genomic variation into account when designing individual or population-wide targeting strategies. VARSCOT is available from https://github.com/BauerLab/VARSCOT .


Assuntos
Sistemas CRISPR-Cas , Biologia Computacional/métodos , Edição de Genes/métodos , Marcação de Genes/métodos , Genômica/métodos , Software , Edição de Genes/normas , Marcação de Genes/normas , Genômica/normas , Internet , Reprodutibilidade dos Testes
8.
Ann Hematol ; 98(8): 1905-1918, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31104089

RESUMO

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/farmacologia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Expressão Gênica , Marcação de Genes/métodos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Lipídeos/administração & dosagem , Lipídeos/química , Camundongos , Camundongos Nus , Nanopartículas/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacocinética , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Blood ; 134(4): 363-373, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31101621

RESUMO

Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and ß are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3ß compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3ß genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3ß binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3ß is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3ß correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.


Assuntos
Quinase 3 da Glicogênio Sintase/genética , Linfoma/etiologia , Terapia de Alvo Molecular , Animais , Biomarcadores Tumorais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Marcação de Genes/métodos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Indóis/farmacologia , Linfoma/diagnóstico , Linfoma/mortalidade , Linfoma/terapia , Maleimidas/farmacologia , Camundongos , Camundongos Transgênicos , Mitose/efeitos dos fármacos , Mitose/genética , Terapia de Alvo Molecular/efeitos adversos , Terapia de Alvo Molecular/métodos , Fuso Acromático/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
10.
BMC Biotechnol ; 19(1): 25, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060546

RESUMO

BACKGROUND: The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined. RESULTS: First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5' side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5' side, indicating unpredictability of the events. CONCLUSIONS: The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.


Assuntos
Sistemas CRISPR-Cas , Embrião de Mamíferos/metabolismo , Edição de Genes/métodos , Marcação de Genes/métodos , Animais , Sequência de Bases , Embrião de Mamíferos/embriologia , Mutação , Análise de Sequência de DNA , Suínos
11.
Cell Mol Life Sci ; 76(20): 4155-4164, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31030226

RESUMO

Evolved xCas9(3.7) variant with broad PAM compatibility has been reported in cell lines, while its editing efficiency was site-specific. Here, we show that xCas9(3.7) can recognize a broad PAMs including NGG, NGA, and NGT, in both embryos and Founder (F0) rabbits. Furthermore, the codon-optimized xCas9-derived base editors, exBE4 and exABE, can dramatically improve the base editing efficiencies in rabbit embryos. Our results demonstrated that the optimized xCas9 with expanded PAM compatibility and enhanced base editing efficiency could be used for precise gene modifications in organisms.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Efeito Fundador , Edição de Genes/métodos , Marcação de Genes/métodos , RNA Guia/genética , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/metabolismo , Códon , Distrofina/genética , Distrofina/metabolismo , Embrião de Mamíferos , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Microinjeções , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , RNA Guia/metabolismo , Coelhos , Repetições de Trinucleotídeos , Zigoto
12.
Plasmid ; 103: 25-35, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30954454

RESUMO

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.


Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Corynebacterium glutamicum/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Marcação de Genes/métodos , Plasmídeos/química , Arginina/biossíntese , Argininossuccinato Liase/genética , Argininossuccinato Liase/metabolismo , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Citrulina/biossíntese , Corynebacterium glutamicum/efeitos dos fármacos , Corynebacterium glutamicum/metabolismo , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Isopropiltiogalactosídeo/farmacologia , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Tetraciclinas/farmacologia
13.
Biomolecules ; 9(4)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018529

RESUMO

The phosphatidylinositol 3-kinase (PI3K) pathway is involved in a myriad of cellular signalling pathways that regulate cell growth, metabolism, proliferation and survival. As a result, alterations in the PI3K pathway are frequently associated with human cancers. Indeed, PIK3CA-the gene encoding the p110α catalytic subunit of PI3K-is one of the most commonly mutated human oncogenes. PIK3CA mutations have also been implicated in non-malignant conditions including congenital overgrowth syndromes and vascular malformations. In order to study the role of PIK3CA mutations in driving tumorigenesis and tissue overgrowth and to test potential therapeutic interventions for these conditions, model systems are essential. In this review we discuss the various mouse models currently available for preclinical studies into the biological consequences and clinical significance of PIK3CA mutations.


Assuntos
Modelos Animais de Doenças , Mutação , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Animais , Marcação de Genes/métodos , Camundongos , Neoplasias/patologia , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo
14.
Methods Cell Biol ; 151: 305-321, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948015

RESUMO

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9) technology enables rapid, targeted, and efficient changes in the genomes of various model organisms. The short guide RNAs (gRNAs) of the CRISPR/Cas9 system can be designed to recognize target DNA within coding regions for functional gene knockouts. Several studies have demonstrated that the CRISPR/Cas9 system efficiently and specifically targets sea urchin genes and results in expected mutant phenotypes. In addition to disrupting gene functions, modifications and additions to the Cas9 protein enable alternative activities targeted to specific sites within the genome. This includes a fusion of cytidine deaminase to Cas9 (Cas9-DA) for single nucleotide conversion in targeted sites. In this chapter, we describe detailed methods for the CRISPR/Cas9 application in sea urchin embryos, including gRNA design, in vitro synthesis of single guide RNA (sgRNA), and the usages of the CRISPR/Cas9 technology for gene knockout and single nucleotide editing. Methods for genotyping the resultant embryos are also provided for assessing efficiencies of gene editing.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Ouriços-do-Mar/genética , Animais , Citidina Desaminase/genética , Marcação de Genes/métodos , Vetores Genéticos , Genoma/genética , Ouriços-do-Mar/crescimento & desenvolvimento
15.
Blood ; 133(26): 2745-2752, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-30975639

RESUMO

Many genetic diseases, including hemophilia, require long-term therapeutic effects. Despite the initial success of liver-directed adeno-associated virus (AAV) gene therapy for hemophilia in clinical trials, long-term sustained therapeutic effects have yet to be seen. One explanation for the gradual decline of efficacy over time is that the nonintegrating AAV vector genome could be lost during cell division during hepatocyte turnover, albeit at a slow pace in adults. Readministering the same vector is challenging as a result of the AAV-neutralizing antibodies elicited by the initial treatment. Here, we investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed gene targeting for sustained treatment of hemophilia B. We developed a donor vector containing a promoterless partial human factor IX (FIX) complementary DNA carrying the hyperactive FIX Padua mutation. A single injection of dual AAV vectors in newborn and adult FIX-knockout (FIX-KO) mice led to stable expression of FIX at or above the normal levels for 8 months. Eight weeks after the vector treatment, we subjected a subgroup of newborn and adult treated FIX-KO mice to a two-thirds partial hepatectomy; all of these animals survived the procedure without any complications or interventions. FIX levels persisted at similar levels for 24 weeks after partial hepatectomy, indicating stable genomic targeting. Our results lend support for the use of a CRISPR/Cas9 approach to achieve lifelong expression of therapeutic proteins.


Assuntos
Sistemas CRISPR-Cas , Fator IX/genética , Marcação de Genes/métodos , Hemofilia B/genética , Hemostasia/genética , Animais , Animais Recém-Nascidos , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Humanos , Camundongos , Camundongos Knockout
16.
Methods Mol Biol ; 1936: 249-274, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820904

RESUMO

Cell-type-specific gene targeting with the Cre/loxP system has become an indispensable technique in experimental neuroscience, particularly for the study of late-born glial cells that make myelin. A plethora of conditional mutants and Cre-expressing mouse lines is now available to the research community that allows laboratories to readily engage in in vivo analyses of oligodendrocytes and their precursor cells. This chapter summarizes concepts and strategies in targeting myelinating glial cells in mice for mutagenesis or imaging, and provides an overview of the most important Cre driver lines successfully used in this rapidly growing field.


Assuntos
Marcação de Genes/métodos , Integrases/metabolismo , Oligodendroglia/citologia , Tamoxifeno/farmacologia , Animais , Linhagem da Célula , Regulação da Expressão Gênica/efeitos dos fármacos , Integrases/genética , Camundongos , Camundongos Transgênicos , Mutagênese , Especificidade de Órgãos , Transgenes
17.
Neuron ; 101(5): 839-862, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30844402

RESUMO

Gene transfer has long been a compelling yet elusive therapeutic modality. First mainly considered for rare inherited disorders, gene therapy may open treatment opportunities for more challenging and complex diseases such as Alzheimer's or Parkinson's disease. Today, examples of striking clinical proof of concept, the first gene therapy drugs coming onto the market, and the emergence of powerful new molecular tools have broadened the number of avenues to target neurological disorders but have also highlighted safety concerns and technology gaps. The vector of choice for many nervous system targets currently is the adeno-associated viral (AAV) vector due to its desirable safety profile and strong neuronal tropism. In aggregate, the clinical success, the preclinical potential, and the technological innovation have made therapeutic AAV drug development a reality, particularly for nervous system disorders. Here, we discuss the rationale, opportunities, limitations, and progress in clinical AAV gene therapy.


Assuntos
Doença de Alzheimer/terapia , Marcação de Genes/métodos , Terapia Genética/métodos , Doença de Parkinson/terapia , Animais , Dependovirus/genética , Marcação de Genes/efeitos adversos , Terapia Genética/efeitos adversos , Humanos
18.
BMC Genomics ; 20(1): 225, 2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890156

RESUMO

BACKGROUND: Large-scale genetic screening using CRISPR-Cas9 technology has emerged as a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biological processes. Here, we report the optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with a temporal control. RESULTS: We designed multiple drug-inducible sgRNA expression vectors and measured their activities using an EGFP gene disruption assay in 11 human and mouse cell lines. The optimal design allows for a tight and inducible control of gene knockout in vitro, and in vivo during a seven-week-long experiment following hematopoietic reconstitution in mice. We next performed parallel genome-wide loss-of-function screens using the inducible and constitutive CRISPR-Cas9 systems. In proliferation-based dropout screens, these two approaches have similar performance in discriminating essential and nonessential genes. In a more challenging phenotypic assay that requires cytokine stimulation and cell staining, we observed similar sensitivity of the constitutive and drug-induced screening approaches in detecting known hits. Importantly, we demonstrate minimal leakiness of our inducible CRISPR screening platforms in the absence of chemical inducers in large-scale settings. CONCLUSIONS: In this study, we have developed a drug-inducible CRISPR-Cas9 system that shows high cleavage efficiency upon induction but low background activity. Using this system, we have achieved inducible gene disruption in a wide range of cell types both in vitro and in vivo. For the first time, we present a systematic side-by-side comparison of constitutive and drug-inducible CRISPR-Cas9 platforms in large-scale functional screens. We demonstrate the tightness and efficiency of our drug-inducible CRISPR-Cas9 system in genome-wide pooled screening. Our design increases the versatility of CRISPR-based genetic screening and represents a significant upgrade on existing functional genomics toolbox.


Assuntos
Sistemas CRISPR-Cas , Carcinoma de Células Renais/genética , Receptores ErbB/antagonistas & inibidores , Marcação de Genes/métodos , Testes Genéticos/métodos , Neoplasias Renais/genética , Inibidores de Proteínas Quinases/farmacologia , Animais , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Proliferação de Células , Células Cultivadas , Receptores ErbB/genética , Genoma , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Camundongos
19.
Artigo em Inglês | MEDLINE | ID: mdl-30921652

RESUMO

Planktonic copepods are a diverse and abundant group of small (~mm sized) aquatic animals that play a critical role in linking the base of the food chain with higher trophic levels. These invertebrates are a primary food source for marine fish larvae. Their ubiquitous presence is thus of vital importance for recruitment of fish stocks and also as promising live feed for finfish production in aquaculture. This paper reviews the application of molecular approaches to understanding copepod physiology, particularly in non-parasitic species. The review includes both targeted gene approaches and untargeted transcriptomic approaches, with suggestions for best practices in each case. Issues particularly relevant to studies of copepods include heterogeneity within species, morphologically cryptic species, experimental artifacts associated with sample handling, and limited annotation of copepod genes and transcripts. The emergence of high-throughput sequencing and associated increased availability of genomic and transcriptomic databases have presented a huge opportunity to advance knowledge of copepod physiology. The research community can leverage this opportunity through efforts to maintain or improve data accessibility, database annotation, and documentation of analytical pipelines.


Assuntos
Copépodes/genética , Transcriptoma , Animais , Copépodes/fisiologia , Perfilação da Expressão Gênica/métodos , Marcação de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos
20.
Biotechnol Adv ; 37(5): 708-729, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30926472

RESUMO

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Marcação de Genes/métodos , RNA/genética , Adenosina Desaminase/genética , Aptâmeros de Nucleotídeos , Corantes Fluorescentes , Hibridização in Situ Fluorescente , Interferência de RNA , Sensibilidade e Especificidade
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