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1.
Arch Virol ; 165(12): 2837-2846, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33025197

RESUMO

Pseudorabies virus (PRV) is a pig pathogen that causes substantial economic losses to the pig industry. Infection of host cells by PRV is mediated by the membrane proteins nectin1 and nectin2, which are presumed to be receptors for PRV infection. Here, we generated nectin1/2 knockout (KO) cells with the aim of establishing a PRV-resistant cell model. Nectin1 and 2 were ablated in PK15 cells by CRISPR/Cas9-mediated gene targeting. PRV infection in either nectin1 or nectin2 KO cells showed a significant reduction in viral growth compared with wild-type (WT) cells. We further simultaneously deleted nectin1 and nectin2 in PK15 cells and found that double KO cells showed no further increase in resistance to PRV compared with single gene-KO cells, despite being more resistant than WT. By investigating the cell entry steps of PRV infection, we found that nectin1 or/and nectin2 KO did not greatly affect virus attachment or internalization to cells but blocked cell-to-cell spread. Our results demonstrate that KO of either nectin1 or nectin2 confers PRV resistance to PK15 cells. This strategy could be applied to establish PRV-resistant pigs with nectin1/2 modifications to benefit the pig industry.


Assuntos
Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/fisiologia , Nectinas/genética , Pseudorraiva/virologia , Animais , Linhagem Celular , Marcação de Genes/métodos , Mutação , Suínos , Doenças dos Suínos/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
2.
Curr Protoc Plant Biol ; 5(3): e20117, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32865887

RESUMO

CRISPR/Cas systems enable gene editing through the induction of site-specific DNA double-strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non-homologous end joining (NHEJ)-mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T-rich regions. Most recently, we tested an improved, temperature-tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants.


Assuntos
Arabidopsis/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes
3.
PLoS One ; 15(9): e0238950, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32960926

RESUMO

Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Edição de Genes/métodos , Marcação de Genes/métodos , Toxinas Biológicas/genética , Alelos , Animais , Sistemas CRISPR-Cas , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Homozigoto , Fenótipo , RNA Guia/genética , Toxinas Biológicas/metabolismo , Transgenes
4.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1659-1671, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924364

RESUMO

To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⁺ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.


Assuntos
Escherichia coli , Inativação Gênica , Marcação de Genes , Técnicas Genéticas , Temperatura , Escherichia coli/genética , Flagelos , Marcação de Genes/métodos , Mutação , Plasmídeos
5.
Nat Commun ; 11(1): 4593, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929070

RESUMO

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.


Assuntos
Alelos , Sistemas CRISPR-Cas/genética , Núcleo Celular/genética , Edição de Genes , Terapia de Substituição Mitocondrial , Animais , Sequência de Bases , Modelos Animais de Doenças , Nanismo/genética , Perda do Embrião/genética , Feminino , Marcação de Genes , Genes Dominantes , Impressão Genômica , Heterozigoto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Reprodutibilidade dos Testes , Fatores de Tempo
6.
Hum Cell ; 33(4): 1142-1154, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32776307

RESUMO

Long noncoding RNAs (lncRNAs) are abnormally expressed in many malignant tumors and involved in regulating the malignant phenotypes of cancer cells. However, the role of LINC00665 in colorectal cancer (CRC) and its regulatory mechanism remain unclear. In this study, real-time polymerase chain reaction (RT-PCR) was used to detect the expressions of LINC00665, miR-9-5p and activating transcription factor 1 (ATF1) mRNA in CRC tissues. The expression of ATF1 in CRC tissues was also detected by immunohistochemistry and Western blot. CCK-8 and colony formation assays were employed to detect cell proliferation. Cell cycle and apoptosis were detected by flow cytometry analysis. Scratch healing assay and Transwell test were exploited to detect cell migration and invasion. The targeting relationships between LINC00665 and miR-9-5p, and miR-9-5p and ATF1 were validated by dual luciferase reporter assay. We found that LINC00665 was significantly overexpressed in CRC tissues, and it was also negatively correlated with the expression of miR-9-5p and positively associated with the expression of ATF1. Besides, LINC00665 promoted the proliferation, migration and invasion of CRC cells, and inhibited cell apoptosis by sponging miR-9-5p. ATF1 was proved to be the downstream target of miR-9-5p and was indirectly regulated by LINC00665. Collectively, it is concluded that LINC00665 contributes to the progression of CRC by regulating miR-9-5p/ATF1 axis.


Assuntos
Fator 1 Ativador da Transcrição/genética , Fator 1 Ativador da Transcrição/metabolismo , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/genética , Expressão Gênica/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/fisiologia , Apoptose/genética , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Colorretais/metabolismo , Progressão da Doença , Marcação de Genes , Humanos , Invasividade Neoplásica/genética , RNA Longo não Codificante/metabolismo
7.
Drug Dev Ind Pharm ; 46(8): 1345-1353, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32643448

RESUMO

PURPOSE: Huashi Baidu formula (HSBDF) was developed to treat the patients with severe COVID-19 in China. The purpose of this study was to explore its active compounds and demonstrate its mechanisms against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through network pharmacology and molecular docking. METHODS: All the components of HSBDF were retrieved from the pharmacology database of TCM system. The genes corresponding to the targets were retrieved using UniProt and GeneCards database. The herb-compound-target network was constructed by Cytoscape. The target protein-protein interaction network was built using STRING database. The core targets of HSBDF were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The main active compounds of HSBDF were docked with SARS-CoV-2 and angiotensin converting enzyme II (ACE2). RESULTS: Compound-target network mainly contained 178 compounds and 272 corresponding targets. Key targets contained MAPK3, MAPK8, TP53, CASP3, IL6, TNF, MAPK1, CCL2, PTGS2, etc. There were 522 GO items in GO enrichment analysis (p < .05) and 168 signaling pathways (p < .05) in KEGG, mainly including TNF signaling pathway, PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, and HIF-1 signaling pathway. The results of molecular docking showed that baicalein and quercetin were the top two compounds of HSBDF, which had high affinity with ACE2. CONCLUSION: Baicalein and quercetin in HSBDF may regulate multiple signaling pathways through ACE2, which might play a therapeutic role on COVID-19.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Simulação de Acoplamento Molecular/métodos , Farmacologia Clínica/métodos , Pneumonia Viral/tratamento farmacológico , Betacoronavirus/química , Betacoronavirus/genética , China , Bases de Dados Factuais , Ontologia Genética , Marcação de Genes , Genes Virais/efeitos dos fármacos , Genes Virais/genética , Humanos , Medicina Tradicional Chinesa , Pandemias , Peptidil Dipeptidase A/efeitos dos fármacos , Peptidil Dipeptidase A/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
8.
Anim Sci J ; 91(1): e13386, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32512638

RESUMO

This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.


Assuntos
Proteína 9 Associada à CRISPR , Eletroporação/métodos , Eletroporação/veterinária , Desenvolvimento Embrionário/genética , Edição de Genes , Marcação de Genes/veterinária , Miostatina/genética , RNA Guia , Suínos/embriologia , Suínos/genética , Zigoto , Animais , Blastocisto , Proteína 9 Associada à CRISPR/farmacologia , Relação Dose-Resposta a Droga
9.
Gene ; 753: 144795, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32450202

RESUMO

The advent of genetic selection and genome modification method assure about a real novel reformation in biotechnology and genetic engineering. With the extensive capabilities of molecular markers of them being stable, cost-effective and easy to use, they ultimately become a potent tool for variety of applications such a gene targeting, selection, editing, functional genomics; mainly for the improvisation of commercially important crops. Three main benefits of molecular marker in the field of agriculture and crop improvement programmes first, reduction of the duration of breeding programmes, second, they allow creation of new genetic variation and genetic diversity of plants and third most promising benefit is help in production of engineered plant for disease resistance, or resistance from pathogen and herbicides. This review is anticipated to present an outline how the techniques have been evolved from the simple conventional applications of DNA based molecular markers to highly throughput CRISPR technology and geared the crop yield. Techniques like using Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems have revolutionised in the field of genome editing. These have been promptly accepted in both the research and commercial industry. On the whole, the widespread use of molecular markers with their types, their appliance in plant breeding along with the advances in genetic selection and genome editing together being a novel strategy to boost crop yield has been reviewed.


Assuntos
Agricultura/métodos , Produtos Agrícolas/genética , Engenharia Genética/métodos , Biomarcadores , Biotecnologia , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Resistência à Doença/genética , Edição de Genes/métodos , Marcação de Genes/métodos , Genoma de Planta/genética , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas/genética
10.
Nat Med ; 26(4): 535-541, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32284612

RESUMO

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and ß-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.


Assuntos
Anemia Falciforme/patologia , Edição de Genes , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras/genética , gama-Globinas/genética , Anemia Falciforme/terapia , Animais , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Estudos de Viabilidade , Feminino , Edição de Genes/métodos , Marcação de Genes/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/patologia , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Cultura Primária de Células , Proteínas Repressoras/metabolismo , Talassemia beta/patologia , Talassemia beta/terapia , gama-Globinas/metabolismo
11.
Nat Biotechnol ; 38(8): 954-961, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32231336

RESUMO

Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Guia/genética , Regulação da Expressão Gênica , Marcação de Genes , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Célula Única , Transcriptoma
12.
J Vet Sci ; 21(2): e26, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32233134

RESUMO

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.


Assuntos
Sistemas CRISPR-Cas , Campylobacter jejuni/genética , Marcação de Genes , Neoplasias Pancreáticas/microbiologia , Streptococcus pyogenes/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos
13.
PLoS One ; 15(3): e0230126, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32226034

RESUMO

The generation of genetically modified mouse models derived from gene targeting (GT) in mouse embryonic stem (ES) cells (mESCs) has greatly advanced both basic and clinical research. Our previous finding that gene targeting at the Myh9 exon2 site in mESCs has a pronounced high homologous recombination (HR) efficiency (>90%) has facilitated the generation of a series of nonmuscle myosin II (NM II) related mouse models. Furthermore, the Myh9 gene locus has been well demonstrated to be a new safe harbor for site-specific insertion of other exogenous genes. In the current study, we intend to investigate the molecular biology underlying for this high HR efficiency from other aspects. Our results confirmed some previously characterized properties and revealed some unreported observations: 1) The comparison and analysis of the targeting events occurring at the Myh9 and several widely used loci for targeting transgenesis, including ColA1, HPRT, ROSA26, and the sequences utilized for generating these targeting constructs, indicated that a total length about 6 kb with approximate 50% GC-content of the 5' and 3' homologous arms, may facilitate a better performance in terms of GT efficiency. 2) Despite increasing the length of the homologous arms, shifting the targeting site from the Myh9 exon2, to intron2, or exon3 led to a gradually reduced GT frequency (91.7, 71.8 and 50.0%, respectively). This finding provides the first evidence that the HR frequency may also be associated with the targeting site even in the same locus. Meanwhile, the decreased trend of the GT efficiency at these targeting sites was consistent with the reduced percentage of simple sequence repeat (SSR) and short interspersed nuclear elements (SINEs) in the sequences for generating the targeting constructs, suggesting the potential effects of these DNA elements on GT efficiency; 3) Our series of targeting experiments and analyses with truncated 5' and 3' arms at the Myh9 exon2 site demonstrated that GT efficiency positively correlates with the total length of the homologous arms (R = 0.7256, p<0.01), confirmed that a 2:1 ratio of the length, a 50% GC-content and the higher amount of SINEs for the 5' and 3' arms may benefit for appreciable GT frequency. Though more investigations are required, the Myh9 gene locus appears to be an ideal location for identifying HR-related cis and trans factors, which in turn provide mechanistic insights and also facilitate the practical application of gene editing.


Assuntos
Marcação de Genes , Recombinação Homóloga/genética , Cadeias Pesadas de Miosina/genética , Animais , Edição de Genes/métodos , Marcação de Genes/métodos , Técnicas de Genotipagem , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas , Transformação Genética
14.
Nat Microbiol ; 5(6): 848-863, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32284562

RESUMO

The most severe form of human malaria is caused by Plasmodium falciparum. Its virulence is closely linked to the increase in rigidity of infected erythrocytes and their adhesion to endothelial receptors, obstructing blood flow to vital organs. Unlike other human-infecting Plasmodium species, P. falciparum exports a family of 18 FIKK serine/threonine kinases into the host cell, suggesting that phosphorylation may modulate erythrocyte modifications. We reveal substantial species-specific phosphorylation of erythrocyte proteins by P. falciparum but not by Plasmodium knowlesi, which does not export FIKK kinases. By conditionally deleting all FIKK kinases combined with large-scale quantitative phosphoproteomics we identified unique phosphorylation fingerprints for each kinase, including phosphosites on parasite virulence factors and host erythrocyte proteins. Despite their non-overlapping target sites, a network analysis revealed that some FIKKs may act in the same pathways. Only the deletion of the non-exported kinase FIKK8 resulted in reduced parasite growth, suggesting the exported FIKKs may instead support functions important for survival in the host. We show that one kinase, FIKK4.1, mediates both rigidification of the erythrocyte cytoskeleton and trafficking of the adhesin and key virulence factor PfEMP1 to the host cell surface. This establishes the FIKK family as important drivers of parasite evolution and malaria pathology.


Assuntos
Eritrócitos/metabolismo , Eritrócitos/parasitologia , Malária/metabolismo , Malária/parasitologia , Fosfotransferases/metabolismo , Plasmodium/fisiologia , Proteínas de Protozoários/metabolismo , Deleção de Genes , Técnicas de Silenciamento de Genes , Marcação de Genes , Humanos , Família Multigênica , Fosfoproteínas , Fosforilação , Fosfotransferases/genética , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Especificidade da Espécie , Virulência
15.
Mar Pollut Bull ; 154: 111038, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32174491

RESUMO

To produce albinism in the marine medaka Oryzias melastigma, we disrupted the solute carrier family 45 (SLC45a2) gene by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with a single guide RNA (sgRNA). Selected sgRNAs were able to target a SLC45a2 gene as confirmed by genotyping and heteroduplex mobility assay (HMA). Of the survived embryos after injection, 54.2% and 60.0% embryos exhibited albinism phenotype by sgRNA1 and sgRNA2, respectively. Deep sequencing at the on-target sites showed different insertion and deletion (indel) mutation profiles near the DNA cleavage sites, indicating high efficacy of producing SLC45a2 knock-out mutants by this method. Moreover, HMA at the potential off-target sites revealed that off-target activity would be induced at a low rate, or not induced at all. This albino marine medaka will be a good model for marine molecular ecotoxicology in establishment of diverse in vivo endpoints, and the application of this efficient gene targeting method in the marine medaka would be useful tool for mechanistic approaches.


Assuntos
Albinismo , Sistemas CRISPR-Cas , Oryzias , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Marcação de Genes
16.
Science ; 368(6488): 290-296, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32217751

RESUMO

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.


Assuntos
Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Marcação de Genes/métodos , Predisposição Genética para Doença , Células HEK293 , Humanos , Mutagênese , Domínios Proteicos , Especificidade por Substrato
17.
Sci Adv ; 6(8): eaay6812, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32128412

RESUMO

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9-mediated "single-cut" genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9-mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.


Assuntos
Sistemas CRISPR-Cas , Dependovirus/genética , Distrofina/genética , Edição de Genes , Terapia Genética , Vetores Genéticos/genética , Distrofia Muscular de Duchenne/genética , Animais , Modelos Animais de Doenças , Éxons , Dosagem de Genes , Expressão Gênica , Marcação de Genes , Técnicas de Transferência de Genes , Camundongos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , Mutação , RNA Guia/genética , Transdução Genética
18.
Biochem Pharmacol ; 175: 113911, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32173365

RESUMO

In response to DNA damage, cell cycle checkpoints produce cell cycle arrest to repair and maintain genomic integrity. Due to the high rates of replication and genetic abnormalities, cancer cells are dependent on replication stress response (RSR) and inhibitors of this pathway are being studied as an anticancer approach. In this direction, we investigated the inhibition of CHK1 and WEE1, key components of RSR, using Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tool. PPRHs designed against WEE1 or CHK1 reduced the viability of different cancer cell lines and showed an increase of apoptosis in HeLa cells. The effect of the PPRHs on cell viability were dose- and time-dependent in HeLa cells. Both the levels of mRNA and protein for each gene were decreased after treatment with the PPRHs. When analyzing the levels of the two CHK1 mRNA splicing variants, CHK1 and CHK1-S, there was a proportional decrease of the two forms, thus maintaining the same expression ratio. PPRHs targeting WEE1 and CHK1 also proved to disrupt cell cycle after 15 h of treatment. Moreover, PPRHs showed a synergy effect when combined with DNA damaging agents, such as methotrexate or 5-Fluorouracil, widely used in clinical practice. This work validates in vitro the usage of PPRHs as a silencing tool against the RSR genes WEE1 and CHK1 and corroborates the potential of inhibiting these targets as a single agent therapy or in combination with other chemotherapy agents in cancer research.


Assuntos
Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem/genética , Inativação Gênica/fisiologia , Marcação de Genes/métodos , Sequências Repetidas Invertidas/fisiologia , Proteínas Tirosina Quinases/genética , Purinas/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Proteínas Tirosina Quinases/antagonistas & inibidores , Purinas/administração & dosagem
19.
PLoS One ; 15(3): e0230566, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32208444

RESUMO

A phenomenon of genetic compensation is commonly observed when an organism with a disease-bearing mutation shows incomplete penetrance of the disease phenotype. Such incomplete phenotypic penetrance, or genetic compensation, is more commonly found in stable knockout models, rather than transient knockdown models. As such, these incidents present a challenge for the disease modeling field, although a deeper understanding of genetic compensation may also hold the key for novel therapeutic interventions. In our study we created a knockout model of slc25a46 gene, which is a recently discovered important player in mitochondrial dynamics, and deleterious mutations in which are known to cause peripheral neuropathy, optic atrophy and cerebellar ataxia. We report a case of genetic compensation in a stable slc25a46 homozygous zebrafish mutant (hereafter referred as "mutant"), in contrast to a penetrant disease phenotype in the first generation (F0) slc25a46 mosaic mutant (hereafter referred as "crispant"), generated with CRISPR/Cas-9 technology. We show that the crispant phenotype is specific and rescuable. By performing mRNA sequencing, we define significant changes in slc25a46 mutant's gene expression profile, which are largely absent in crispants. We find that among the most significantly altered mRNAs, anxa6 gene stands out as a functionally relevant player in mitochondrial dynamics. We also find that our genetic compensation case does not arise from mechanisms driven by mutant mRNA decay. Our study contributes to the growing evidence of the genetic compensation phenomenon and presents novel insights about Slc25a46 function. Furthermore, our study provides the evidence for the efficiency of F0 CRISPR screens for disease candidate genes, which may be used to advance the field of functional genetics.


Assuntos
Sistemas CRISPR-Cas , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Ataxia Cerebelar/genética , Modelos Animais de Doenças , Feminino , Marcação de Genes , Masculino , Mutagênese , Mutação , Atrofia Óptica/genética , Doenças do Sistema Nervoso Periférico/genética
20.
Int J Mol Sci ; 21(3)2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32046108

RESUMO

Interleukin-27 (IL-27) has shown promise in halting tumor growth and mediating tumor regression in several models, including prostate cancer. We describe our findings on the effects of IL-27 on the gene expression changes of TC2R prostate adenocarcinoma cells. We utilized RNAseq to assess profile differences between empty vector control, vector delivering IL-27 modified at its C-terminus with a non-specific peptide, and IL-27 modified at the C-terminus with a peptide targeting the IL-6-Rα. The targeted IL-27 had higher bioactivity and activity in vivo in a recent study by our group, but the mechanisms underlying this effect had not been characterized in detail at the gene expression level on tumor cells. In the present work, we sought to examine potential mechanisms for targeted IL-27 enhanced activity directly on tumor cells. The targeted IL-27 appeared to modulate several changes that would be consistent with an anti-tumor effect, including upregulation in the Interferon (IFN) and Interferon regulatory factor (IRF), oxidative phosphorylation, Janus kinase/Signal transducers and activators of transcription (JAK/STAT), and eukaryotic initiation factor 2 (EIF2) signaling. Of these signaling changes predicted by ingenuity pathway analyses (IPA), the novel form also with the highest significance (-log(Benjamini-Hochberg (B-H)) p-value) was the EIF2 signaling upregulation. We validated this predicted change by assaying for eukaryotic initiation factor 2 alpha (eIF2α), or phosphorylated eIF2α (p-eIF2α), and caspase-3 levels. We detected an increase in the phosphorylated form of eIF2α and in the cleaved caspase-3 fraction, indicating that the EIF2 signaling pathway was upregulated in these prostate tumor cells following targeted IL-27 gene delivery. This approach of targeting cytokines to enhance their activity against cancer cells is a novel approach to help augment IL-27's bioactivity and efficacy against prostate tumors and could be extended to other conditions where it could help interfere with the EIF2α pathway and promote caspase-3 activation.


Assuntos
Adenocarcinoma/metabolismo , Marcação de Genes/métodos , Terapia Genética/métodos , Interleucina-27/genética , Neoplasias da Próstata/metabolismo , Receptores de Interleucina-6/genética , Transdução de Sinais , Adenocarcinoma/genética , Animais , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/metabolismo , Interferons/metabolismo , Interleucina-27/química , Interleucina-27/metabolismo , Janus Quinases/metabolismo , Masculino , Camundongos , Neoplasias da Próstata/genética , Domínios Proteicos , Receptores de Interleucina-6/metabolismo , Fatores de Transcrição STAT/metabolismo
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