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1.
Cancer Sci ; 110(10): 3061-3067, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31444833

RESUMO

Tryptophan metabolism is important to induce immune tolerance in tumors. To date, 3 types of tryptophan-metabolizing enzymes have been identified: indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and IDO2) and tryptophan 2,3-dioxygenase 2. Numerous studies have focused on IDO1 as its expression is enhanced in various cancers. Recently, IDO2 has been identified as a tryptophan-metabolizing enzyme that is involved in several immune functions and expressed in cancers such as pancreatic cancer. However, the biological role of IDO2 in the induction of immune tolerance in tumors has not yet been reported. In the present study, we examined the effects of Ido2 depletion on tumor growth in a mouse model of Lewis lung carcinoma by using Ido2-knockout mice. Ido2-knockout mice had reduced tumor volumes compared to WT mice. Furthermore, Ido2 depletion altered the tumor microenvironment, such as tryptophan accumulation and kynurenine reduction, leading to enhancement of immune cell invasion. Finally, enzyme-linked immunospot assay revealed that Ido2 depletion enhanced γ-interferon secretion in the tumor. In conclusion, Ido2 is an important immune regulator in the tumor microenvironment. Our data indicate that IDO2 is a potential target for cancer treatment and drug development.


Assuntos
Carcinoma Pulmonar de Lewis/terapia , Técnicas de Inativação de Genes/métodos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interferon gama/metabolismo , Animais , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Cinurenina/metabolismo , Masculino , Camundongos , Triptofano/metabolismo , Carga Tumoral , Evasão Tumoral , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
2.
World J Microbiol Biotechnol ; 35(7): 111, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280424

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Edição de Genes/métodos , Regulação Fúngica da Expressão Gênica , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Saccharomyces cerevisiae/genética
3.
Life Sci ; 231: 116586, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31220528

RESUMO

AIMS: Lipocalin 2 (Lcn2/NGAL) belongs to lipocalin superfamily with diverse functions. The precise function of Lcn2, particularly in cancer development, remains to be elucidated yet. In an attempt to knockout of Lcn2 expression by CRISPR/Cas 9 technology in a highly aggressive and invasive prostate cancer cell line and to evaluate the combination therapy with cisplatin (CDDP), this study was conducted. MAIN METHODS: Control CRISPR/Cas9 plasmid and homology-directed repair plasmid or validated human Lcn2 CRISPR/Cas9 KO plasmids were co-transfected into PC3 cells using fugene HD transfection reagent. The stable cells were selected in the presence of puromycin. Correspondingly, knock out of Lcn2 was evaluated by RT-PCR, ELISA, and immunocytochemistry. PC3-Scr (control) and Lcn2-KO (PC3 cells in which lcn2 has been knocked out) were treated with or without cisplatin (CDDP). Cell proliferative ability was measured by WST-1 and colony-formation assays. Apoptosis was evaluated by DAPI staining, in situ cell death detection (TUNEL) assay, and cell death detection ELISA plus methods. The migration capabilities were studied by wound healing/scratch and transwell assays. KEY FINDINGS: Lcn2 knock out in a highly aggressive and invasive cancer cell like PC3 decreased cell proliferation and increased the sensitivity of CDDP. Conspicuously, loss of Lcn2 expression effectively enhanced CDDP-induced apoptosis in PC3 cells. Lcn2 knock out by CRISPR/Cas9 technology decreased the cell migration capacity of PC3 cells as well. SIGNIFICANCE: Lcn2 not only is a valuable and useful biomarker for diagnosis and prognosis of prostate cancer but also and more importantly is a potential novel emerging therapeutic target.


Assuntos
Cisplatino/farmacologia , Lipocalina-2/genética , Lipocalina-2/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Inativação de Genes/métodos , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transdução de Sinais
4.
Methods Mol Biol ; 1971: 189-210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980304

RESUMO

Postgenomic analyses of Leishmania biology benefit from rapid and precise methods for gene manipulation. Traditional methods of gene knockout or tagging by homologous recombination have limitations: they tend to be slow and require successive transfection and selection rounds to knock out multiple alleles of a gene. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems overcome these limitations. We describe here in detail a simple, rapid, and scalable method for CRISPR-Cas9-mediated gene knockout and tagging in Leishmania. This method details how to use simple PCR to generate (1) templates for single guide RNA (sgRNA) transcription in cells expressing Cas9 and T7 RNA polymerase and (2) drug-selectable editing cassettes, using a modular set of plasmids as templates. pT plasmids allow for amplification of drug resistance genes for knockouts and pPLOT plasmids provide a choice of different tags to generate N- or C-terminally tagged proteins. We describe how to use an online platform ( LeishGEdit.net ) for automated primer design and how to perform PCRs and transfections in small batches or on 96-well plates for large-scale knockout or tagging screens. This method allows generation of knockout mutants or tagged cell lines within 1 week.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Leishmania/genética , Recombinação Homóloga , Plasmídeos/genética , Transfecção
5.
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
6.
Nat Methods ; 16(5): 405-408, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962622

RESUMO

Systematic investigation of the genetic interactions that influence metastatic potential has been challenging. Here we developed massively parallel CRISPR-Cpf1/Cas12a crRNA array profiling (MCAP), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library of 11,934 arrays targeting 325 pairwise combinations of genes implicated in metastasis. By assessing the metastatic potential of the double knockouts in mice, we unveiled a quantitative landscape of genetic interactions that drive metastasis.


Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endonucleases/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Metástase Neoplásica/genética , Animais , Proteína 9 Associada à CRISPR/genética , Linhagem Celular Tumoral , Camundongos , Análise de Sequência de RNA
7.
Fungal Biol ; 123(4): 274-282, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30928036

RESUMO

The fungus Purpureocillium lavendulum (formally Paecilomyces lilacinus) is a natural enemy of insects and plant-parasitic nematodes, and has been used as an important bio-control agent against agricultural pests all over the world. In order to understand the genetic mechanisms governing its biocontrol efficiency and other biological processes, an effective gene disruption system is needed. Here we report the development of an efficient system which integrates selective markers that differ from Purpureocillium lilacinum, a one-step construction method for gene knockout plasmids, and a ku80 knockout strain for efficient homologous recombination. With this system, we effectively disrupted the transcription factors in the central regulation pathway of sporulation and a serine protease which were contributed to nematode infection, demonstrating this system as an efficient gene disrupting system for further characterization of genes involved in the development and pathogenesis of this fungus.


Assuntos
Técnicas de Inativação de Genes/métodos , Genética Microbiana/métodos , Hypocreales/genética , Biologia Molecular/métodos , Vetores Genéticos , Recombinação Homóloga , Plasmídeos , Seleção Genética
8.
Methods Mol Biol ; 1961: 185-209, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912047

RESUMO

The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based OPTimized inducible gene KnockOut method (OPTiKO) for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single guide RNAs (sgRNAs) that drive Cas9 activity. In order to ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes/métodos , Humanos , Células-Tronco Pluripotentes/metabolismo , RNA Guia/genética
9.
Methods Mol Biol ; 1953: 213-230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912024

RESUMO

The use of CRISPR/Cas9 to modify the mouse genome has gained immense interest in the past few years since it allows the direct modification of embryos, bypassing the need of labor-intensive procedures for the manipulation of embryonic stem cells. By shortening the overall timelines and reducing the costs for the generation of new genetically modified mouse lines (Li et al., Nat Biotechnol 31: 681-683, 2013), this technology has rapidly become a major tool for in vivo drug discovery applications.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Camundongos/genética , Alelos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Genoma , Técnicas de Genotipagem/métodos , Humanos , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , RNA Guia/genética
10.
Mol Biol Cell ; 30(10): 1198-1213, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30865555

RESUMO

Mitochondria are essential and dynamic organelles undergoing constant fission and fusion. The primary players in mitochondrial morphology (MFN1/2, OPA1, DRP1) have been identified, but their mechanism(s) of regulation are still being elucidated. ARL2 is a regulatory GTPase that has previously been shown to play a role in the regulation of mitochondrial morphology. Here we demonstrate that ELMOD2, an ARL2 GTPase-activating protein (GAP), is necessary for ARL2 to promote mitochondrial elongation. We show that loss of ELMOD2 causes mitochondrial fragmentation and a lower rate of mitochondrial fusion, while ELMOD2 overexpression promotes mitochondrial tubulation and increases the rate of fusion in a mitofusin-dependent manner. We also show that a mutant of ELMOD2 lacking GAP activity is capable of promoting fusion, suggesting that ELMOD2 does not need GAP activity to influence mitochondrial morphology. Finally, we show that ELMOD2, ARL2, Mitofusins 1 and 2, Miros 1 and 2, and mitochondrial phospholipase D (mitoPLD) all localize to discrete, regularly spaced puncta along mitochondria. These results suggest that ELMOD2 is functioning as an effector downstream of ARL2 and upstream of the mitofusins to promote mitochondrial fusion. Our data provide insights into the pathway by which mitochondrial fusion is regulated in the cell.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Dinâmica Mitocondrial/fisiologia , Animais , Células COS , Linhagem Celular , Cercopithecus aethiops , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes/métodos , Humanos , Fusão de Membrana/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo
11.
Methods Mol Biol ; 1955: 61-76, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30868519

RESUMO

The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for gene knockout in T. cruzi, later on for endogenous gene tagging and more recently for gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have generated homogeneous populations of genetically modified parasites in 4-5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the gene. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines generated by CRISPR/Cas9, and C-terminal tagging of endogenous genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Trypanosoma cruzi/genética , Doença de Chagas/parasitologia , Reparo do DNA , Humanos , Transfecção/métodos
12.
Methods Mol Biol ; 1940: 77-95, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788819

RESUMO

Efficient and reliable methods for gene editing are critical for the generation of loss-of-gene function stem cells and genetically modified mice. Here, we outline the application of CRISPR-Cas9 technology for gene editing in mouse embryonic stem cells (mESCs) to generate knockout ESC chimeras for the fast-tracked analysis of gene function. Furthermore, we describe the application of gene editing directly to mouse epiblast stem cells (mEpiSCs) for modelling germ layer differentiation in vitro.


Assuntos
Mutação da Fase de Leitura/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Camadas Germinativas/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Sistemas CRISPR-Cas/genética , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Camundongos , Plasmídeos/genética , RNA Guia/genética
13.
DNA Cell Biol ; 38(4): 314-321, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30762444

RESUMO

The shortage of human hepatocytes continues to be a significant limitation for the widespread application of hepatocyte transplantation and bioartificial liver (BAL) support therapy. Recombinant activation gene 2 (Rag2) and fumarylacetoacetate hydrolase (Fah)-deficient mice could be highly repopulated with human hepatocytes. However, Fah/Rag2-deficient mice can only produce up to 1 × 108 human hepatocytes per mouse. We hypothesized that 2-10 × 1010 human hepatocytes can be produced per Fah/Rag2-deficient pig, which is an adequate supply for hepatocyte transplantation and BAL therapy. In a novel approach, we used stably transfected Cas9 cells and single-guide RNA adenoviruses containing fluorescent reporters to enrich porcine cells with Fah/Rag2 dual gene mutations. This resulted in the construction of Fah/Rag2 double knockout porcine iliac artery endothelial cells, which were subsequently used for generating Fah/Rag2-deficient pigs.


Assuntos
Adenoviridae/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes/métodos , Hidrolases/deficiência , Hidrolases/genética , Animais , Sequência de Bases , Linhagem Celular , Mutação , Suínos , Fatores de Tempo
14.
Interdiscip Sci ; 11(1): 33-44, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30758766

RESUMO

In recent years, metabolic engineering has gained central attention in numerous fields of science because of its capability to manipulate metabolic pathways in enhancing the expression of target phenotypes. Due to this, many computational approaches that perform genetic manipulation have been developed in the computational biology field. In metabolic engineering, conventional methods have been utilized to upgrade the generation of lactate and succinate in E. coli, although the yields produced are usually way below their theoretical maxima. To overcome the drawbacks  of such conventional methods, development of hybrid algorithm is introduced to obtain an optimal solution by proposing a gene knockout strategy in E. coli which is able to improve the production of lactate and succinate. The objective function of the hybrid algorithm is optimized using a swarm intelligence optimization algorithm and a Simple Constrained Artificial Bee Colony (SCABC) algorithm. The results maximize the production of lactate and succinate by resembling the gene knockout in E. coli. The Flux Balance Analysis (FBA) is integrated in a hybrid algorithm to evaluate the growth rate of E. coli as well as the productions of lactate and succinate. This results in the identification of a gene knockout list that contributes to maximizing the production of lactate and succinate in E. coli.


Assuntos
Escherichia coli/genética , Técnicas de Inativação de Genes/métodos , Ácido Láctico/metabolismo , Redes e Vias Metabólicas/fisiologia , Ácido Succínico/metabolismo , Algoritmos , Simulação por Computador , Modelos Biológicos
15.
Methods Mol Biol ; 1925: 163-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30674026

RESUMO

The store-operated calcium (Ca2+) entry (SOCE) pathway is an essential Ca2+ signaling pathway in non-excitable cells that serve many physiological functions. SOCE is mediated through the plasma membrane (PM) protein, Orai1, and the endoplasmic reticulum protein, stromal interaction molecule 1 (STIM1). One of the most well-established methods to study SOCE is using the Ca2+-sensing dye, fura-2. Here we describe a detailed protocol on how to use fura-2 to study Ca2+ signaling from SOCE in human embryonic kidney (HEK) cells.


Assuntos
Cálcio/análise , Corantes Fluorescentes/química , Fura-2/química , Imagem Óptica/métodos , Cálcio/metabolismo , Sinalização do Cálcio , Cátions Bivalentes/análise , Cátions Bivalentes/metabolismo , Técnicas de Inativação de Genes/métodos , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/genética , Molécula 2 de Interação Estromal/metabolismo , Transfecção/métodos
16.
BMC Biotechnol ; 19(1): 1, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30606176

RESUMO

BACKGROUND: Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developing genetic engineering tools for dogs. In this study, we attempted to generate optimized CRISPR/Cas9 system to target canine tumor protein 53 (TP53), one of the most crucial tumor suppressor genes, to establish TP53 knockout canine cells for canine cancer research. RESULTS: We constructed CRISPR/Cas9 vectors using each of three TP53 gene-targeting guide RNAs (gRNAs) with minimal off-target potential. After transfection, we obtained several clones of TP53 knockout cells containing "indel" mutations in the targeted locus which had infinite cellular life span, resistance to genotoxicity, and unstable genomic status in contrast to normal cells. Of the established TP53 knockout cells, TP53KO#30 cells targeted by TP53 gRNA #30 showed non-cancerous phenotypes without oncogenic activation both in vitro and in vivo. More importantly, no off-target alteration was detected in TP53KO#30 cells. We also tested the developmental capacity of TP53 knockout cells after application of the somatic cell nuclear transfer technique. CONCLUSIONS: Our results indicated that TP53 in canine cells was effectively and specifically targeted by our CRISPR/Cas9 system. Thus, we suggest our CRISPR/Cas9-derived canine TP53 knockout cells as a useful platform to reveal novel oncogenic functions and effects of developing anti-cancer therapeutics.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Genes p53 , Neoplasias/genética , Neoplasias/veterinária , Animais , Animais Geneticamente Modificados , Cães , Fibroblastos/fisiologia , Masculino , Neoplasias/prevenção & controle
17.
Plant Cell Rep ; 38(4): 487-501, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30684023

RESUMO

KEY MESSAGE: The analysis of 93 mutant alleles in 18 genes demonstrated that CRISPR-Cas9 is a robust tool for targeted mutagenesis in maize, permitting efficient generation of single and multiple knockouts. CRISPR-Cas9 technology is a simple and efficient tool for targeted mutagenesis of the genome. It has been implemented in many plant species, including crops such as maize. Here we report single- and multiple-gene mutagenesis via stably transformed maize plants. Two different CRISPR-Cas9 vectors were used allowing the expression of multiple guide RNAs and different strategies to knockout either independent or paralogous genes. A total of 12 plasmids, representing 28 different single guide RNAs (sgRNAs), were generated to target 20 genes. For 18 of these genes, at least one mutant allele was obtained, while two genes were recalcitrant to sequence editing. 19% (16/83) of mutant plants showed biallelic mutations. Small insertions or deletions of less than ten nucleotides were most frequently observed, regardless of whether the gene was targeted by one or more sgRNAs. Deletions of defined regions located between the target sites of two guide RNAs were also reported although the exact deletion size was variable. Double and triple mutants were created in a single step, which is especially valuable for functional analysis of genes with strong genetic linkage. Off-target effects were theoretically limited due to rigorous sgRNA design and random experimental checks at three potential off-target sites did not reveal any editing. Sanger chromatograms allowed to unambiguously class the primary transformants; the majority (85%) were fully edited plants transmitting systematically all detected mutations to the next generation, generally following Mendelian segregation.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes/métodos , Zea mays/genética , Edição de Genes , Genoma de Planta/genética , Mutagênese/genética
18.
Anim Sci J ; 90(3): 366-371, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30623527

RESUMO

Porcine viral diarrhea is an acute and highly contagious enteric disease in pigs which causes huge economic losses in pig industry worldwide. Transmissible gastroenteritis virus (TGEV) is main pathogens responsible for piglets viral diarrhea. Knockout the host cellular surface receptor for TGEV may be an effective way to accelerate the breeding of resistant pigs. In this study, we applied site-specific editing pAPN which is effective in swine testis (ST) cells. Site-specific editing of pAPN reduced TGEV proliferation in ST cells by 96%-99% at different time periods post-infection. Next, the site-specific editing of pAPN porcine fetal fibroblasts were produced, and then the cell colonies were used as donor cells to generate the site-specific editing of pAPN pigs. Our research findings will not only offer a more thorough understanding of the pathogenesis of piglet diarrhea and lay the foundation for breeding TGEV-resistant piglets, but also understanding the molecular mechanisms involved in coronaviral infections.


Assuntos
Cruzamento/métodos , Antígenos CD13/genética , Diarreia/prevenção & controle , Diarreia/veterinária , Gastroenterite Suína Transmissível/prevenção & controle , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Receptores Virais/genética , Doenças dos Suínos/prevenção & controle , Suínos , Animais , Diarreia/virologia , Fibroblastos/enzimologia , Gastroenterite Suína Transmissível/virologia , Masculino , Doenças dos Suínos/virologia , Testículo/citologia , Testículo/virologia , Vírus da Gastroenterite Transmissível/patogenicidade , Vírus da Gastroenterite Transmissível/fisiologia , Replicação Viral
19.
Cell Mol Life Sci ; 76(9): 1697-1711, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30694346

RESUMO

Biologic function of the majority of microRNAs (miRNAs) is still unknown. Uncovering the function of miRNAs is hurdled by redundancy among different miRNAs. The deletion of Dgcr8 leads to the deficiency in producing all canonical miRNAs, therefore, overcoming the redundancy issue. Dgcr8 knockout strategy has been instrumental in understanding the function of miRNAs in a variety of cells in vitro and in vivo. In this review, we will first give a brief introduction about miRNAs, miRNA biogenesis pathway and the role of Dgcr8 in miRNA biogenesis. We will then summarize studies performed with Dgcr8 knockout cell models with a focus on embryonic stem cells. After that, we will summarize results from various in vivo Dgcr8 knockout models. Given significant phenotypic differences in various tissues between Dgcr8 and Dicer knockout, we will also briefly review current progresses on understanding miRNA-independent functions of miRNA biogenesis factors. Finally, we will discuss the potential use of a new strategy to stably express miRNAs in Dgcr8 knockout cells. In future, Dgcr8 knockout approaches coupled with innovations in miRNA rescue strategy may provide further insights into miRNA functions in vitro and in vivo.


Assuntos
Processamento Alternativo/genética , RNA Helicases DEAD-box/genética , Células-Tronco Embrionárias/citologia , Técnicas de Inativação de Genes/métodos , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Animais , Deleção de Genes , Humanos , Camundongos , Células-Tronco Neurais/citologia
20.
PLoS Negl Trop Dis ; 13(1): e0007088, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30640901

RESUMO

Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.


Assuntos
Expressão Gênica , Técnicas de Inativação de Genes/métodos , Genética Microbiana/métodos , Biologia Molecular/métodos , Testes de Sensibilidade Parasitária/métodos , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Antiprotozoários/farmacologia , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Eletroporação , Nifurtimox/farmacologia , Nitroimidazóis/farmacologia , Trypanosoma cruzi/crescimento & desenvolvimento
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