Programmed mutation of liver fluke granulin using CRISPR/Cas9 attenuates virulence of infection-induced hepatobiliary morbidity

AbstractInfections with several flatworm parasites represent group 1 biological carcinogens, i.e. definite causes of cancer. Infection with the food-borne liver fluke Opisthorchis viverrini causes cholangiocarcinoma (CCA). Whereas the causative agent for most cancers, including CCA in the West, remains obscure, the principal risk factor for CCA in Thailand is opisthorchiasis. We exploited this established link to explore the role of the secreted parasite growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions of the biliary tract. We targeted the Ov-grn-1 gene for programmed knockout and investigated gene-edited parasites in vitro and in experimentally infected hamsters. Both adult and juvenile stages of the liver fluke were transfected with a plasmid encoding a guide RNA sequence specific for exon 1 of Ov-grn-1 and the Cas9 nuclease. Deep sequencing of amplicon libraries from genomic DNA from gene-edited parasites exhibited programmed, Cas9-catalyzed mutations within the Ov-grn-1 locus, and tandem analyses by RT-PCR and western blot revealed rapid depletion of Ov-grn-1 transcripts and protein. Newly excysted juvenile flukes that had undergone editing of Ov-grn-1 colonized the biliary tract, grew and developed over a period of 60 days, were active and motile, and induced a clinically relevant pathophysiological tissue phenotype of attenuated biliary hyperplasia and fibrosis in comparison to infection with wild type flukes. This is the first report of gene knock-out using CRISPR/Cas9 in a parasitic flatworm, demonstrating the activity and utility of the process for functional genomics in these pathogens. The striking clinical phenotype highlights the role in virulence that liver fluke growth factors play in biliary tract morbidity during chronic opisthorchiasis.

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In vitro and in vivo trematode models for chemotherapeutic studies

Parasitology ◽

10.1017/s0031182009991739 ◽

2009 ◽

Vol 137 (3) ◽

pp. 589-603 ◽

Cited By ~ 100

Author(s):

J. KEISER

Keyword(s):

Liver Fluke ◽

Fasciola Hepatica ◽

State Of The Art ◽

Parasitic Diseases ◽

Current State ◽

Food Borne ◽

Essential Components ◽

Discovery Pipeline

SUMMARYSchistosomiasis and food-borne trematodiases are chronic parasitic diseases affecting millions of people mostly in the developing world. Additional drugs should be developed as only few drugs are available for treatment and drug resistance might emerge. In vitro and in vivo whole parasite screens represent essential components of the trematodicidal drug discovery cascade. This review describes the current state-of-the-art of in vitro and in vivo screening systems of the blood fluke Schistosoma mansoni, the liver fluke Fasciola hepatica and the intestinal fluke Echinostoma caproni. Examples of in vitro and in vivo evaluation of compounds for activity are presented. To boost the discovery pipeline for these diseases there is a need to develop validated, robust high-throughput in vitro systems with simple readouts.

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Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1703718114 ◽

2017 ◽

Vol 114 (31) ◽

pp. 8283-8288 ◽

Cited By ~ 24

Author(s):

Jun Liu ◽

Yong Juan Zhao ◽

Wan Hua Li ◽

Yun Nan Hou ◽

Ting Li ◽

...

Keyword(s):

Catalytic Domain ◽

Bimolecular Fluorescence Complementation ◽

Knock Out ◽

Guide Rna ◽

Type Iii ◽

Cyclic Adp Ribose ◽

Human Multiple Myeloma ◽

Hybrid Screening

CD38 catalyzes the synthesis of the Ca2+ messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca2+ stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells. Because type III CD38 is topologically amenable to cytosolic regulation, we used yeast-two-hybrid screening to identify cytosolic Ca2+ and integrin-binding protein 1 (CIB1), as its interacting partner. The results from immunoprecipitation, ELISA, and bimolecular fluorescence complementation confirmed that CIB1 binds specifically to the catalytic domain of CD38, in vivo and in vitro. Mutational studies established that the N terminus of CIB1 is the interacting domain. Using shRNA to knock down and Cas9/guide RNA to knock out CIB1, a direct correlation between the cellular cADPR and CIB1 levels was demonstrated. The results indicate that the type III CD38 is functionally active in producing cellular cADPR and that the activity is specifically modulated through interaction with cytosolic CIB1.

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In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

10.21926/obm.genet.2001123 ◽

2021 ◽

Vol 5 (1) ◽

Author(s):

Masahiro Sato ◽

◽

Hansol Jin ◽

Eri Akasaka ◽

Kazuchika Miyoshi

Keyword(s):

Viral Infection ◽

Target Gene ◽

Direct Injection ◽

Porcine Genome ◽

Knock Out ◽

Guide Rna ◽

Insertion And Deletion ◽

Cas9 Protein ◽

Cytoplasmic Injection

The production of genetically modified (GM) pigs is considered valuable in biomedical research for the development of model animals for various diseases and pigs with resistance against viral infection. The porcine genome may be modified using several methods, such as somatic cell nuclear transfer (SCNT) using GM cells as the SCNT donor, direct injection of the transgene or the genome editing components (GEC) into fertilized eggs referred to as zygotes, the in vitro electroporation (EP) of the zygotes in the presence of GECs, viral infection using retroviruses, injection of the GECs into the SCNT-treated embryos, and the in vitro EP of the SCNT-treated embryos in the presence of GECs. In our previous study, we administered a cytoplasmic injection of CRISPR/Cas9-based GEC into parthenogenetically-activated porcine oocytes (referred to as parthenotes) and observed that these oocytes comprised a mixture of genome-edited and genome-unedited cells, referred to as the “mosaic”. In contrast, when in vitro EP of the SCNT-treated embryos in the presence of GEC was performed, bi-allelic knock out (KO) of the target gene was detected in most oocytes (82%; 9/11). The production of bi-allelic KO piglets is particularly beneficial for investigating GM domestic animals as it does not require further breeding trials to obtain bi-allelic KO individuals, which would otherwise be a time-consuming and laborious task. In this context, the present study was aimed to confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos without multiple breeding trials, for which parthenotes were subjected to EP in the presence of a ribonucleoprotein containing Cas9 protein and single-guide RNA (targeted toward GGTA1). The treated embryos were cultured until they transformed into blastocysts. The genomic DNA isolated from these blastocysts was used for molecular biology analysis to detect the possible insertion and deletion of sequences (indels) at the GGTA1 locus. Among the 32 blastocysts obtained, 21 (66%) were observed to be the bi-allelic KO ones. The remaining embryos either had a normal phenotype (25%; 8/32) or mosaic mutations (9%; 3/32). These findings confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos.

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Activity of tribendimidine and praziquantel combination therapy against the liver fluke Opisthorchis viverriniin vitro and in vivo

Journal of Helminthology ◽

10.1017/s0022149x12000387 ◽

2012 ◽

Vol 87 (2) ◽

pp. 252-256 ◽

Cited By ~ 11

Author(s):

J. Keiser ◽

R. Adelfio ◽

M. Vargas ◽

P. Odermatt ◽

S. Tesana

Keyword(s):

Worm Burden ◽

Liver Fluke ◽

Combination Index ◽

Public Health Problem ◽

Important Public Health Problem ◽

Important Public Health ◽

Single Drug ◽

Food Borne

AbstractOpisthorchiasis, caused by the liver fluke Opisthorchis viverrini, a food-borne trematode, is an important public health problem; however, only a single drug, praziquantel is available. We investigated tribendimidine–praziquantel combinations against O. viverriniin vitro and in vivo. The IC50 values of 0.16 μg/ml and 0.05 μg/ml were determined for praziquantel and tribendimidine, respectively, against adult O. viverriniin vitro. When O. viverrini was exposed to both drugs simultaneously (using a drug ratio based on the IC50 (1:3.2)) a synergistic effect was calculated (combination index (CI) at the IC50= 0.7). A similar result was observed when drug addition in vitro was spaced by the respective half-lives of the drugs (a CI of 0.78 at the IC50 for tribendimidine followed by praziquantel and a CI of 0.47 at the IC50 for praziquantel followed by tribendimidine). In vivo median-effect dose (ED50) values of 191 mg/kg and 147 mg/kg were calculated for praziquantel and tribendimidine, respectively. Low to moderate worm burden reductions (38–62%) were observed in O. viverrini infected hamsters when both drugs were administered simultaneously or on subsequent days, pointing to antagonistic effects in vivo. Further studies are necessary to understand the striking differences between the in vitro and in vivo observations using combinations of praziquantel and tribendimidine on O. viverrini.

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In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

10.21926/obm.genet.2101123 ◽

2021 ◽

Vol 5 (1) ◽

Author(s):

Masahiro Sato ◽

◽

Hansol Jin ◽

Eri Akasaka ◽

Kazuchika Miyoshi

Keyword(s):

Viral Infection ◽

Target Gene ◽

Direct Injection ◽

Porcine Genome ◽

Knock Out ◽

Guide Rna ◽

Insertion And Deletion ◽

Cas9 Protein ◽

Cytoplasmic Injection

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Staphylococcus aureusCas9 is a multiple-turnover enzyme

10.1101/340042 ◽

2018 ◽

Cited By ~ 1

Author(s):

Paul Yourik ◽

Ryan T. Fuchs ◽

Megumu Mabuchi ◽

Jennifer L. Curcuru ◽

G. Brett Robb

Keyword(s):

Dna Cleavage ◽

Great Promise ◽

Guide Rna ◽

Future Design ◽

Cas9 Nuclease ◽

A Genome ◽

Rate Of Reaction ◽

High Degree ◽

Adaptive Immune Systems

ABSTRACTCas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences.S. aureus(SauCas9) and especiallyS. pyogenes(SpyCas9) are among the best-characterized Cas9 proteins and share about 17% sequence identity. A notable feature of SpyCas9 is an extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. Usingin vitrobiochemistry and enzyme kinetics we directly compare SpyCas9 and SauCas9 activities. Here, we report that in contrast to SpyCas9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-stranded break observed previously with SpyCas9, thus providing new insights and considerations for future design of CRISPR/Cas9-based applications.

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Multiplexed tat-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape

Viruses ◽

10.3390/v12111223 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1223

Author(s):

Youdiil Ophinni ◽

Sayaka Miki ◽

Yoshitake Hayashi ◽

Masanori Kameoka

Keyword(s):

T Cells ◽

Viral Escape ◽

Escape Mutant ◽

Knock Out ◽

Guide Rna ◽

Acute Hiv ◽

Almost All ◽

First Time ◽

Hiv 1

HIV-1 cure strategy by means of proviral knock-out using CRISPR-Cas9 has been hampered by the emergence of viral resistance against the targeting guide RNA (gRNA). Here, we proposed multiple, concentrated gRNA attacks against HIV-1 regulatory genes to block viral escape. The T cell line were transduced with single and multiple gRNAs targeting HIV-1 tat and rev using lentiviral-based CRISPR-Cas9, followed by replicative HIV-1NL4-3 challenge in vitro. Viral p24 rebound was observed for almost all gRNAs, but multiplexing three tat-targeting gRNAs maintained p24 suppression and cell viability, indicating the inhibition of viral escape. Multiplexed tat gRNAs inhibited acute viral replication in the 2nd round of infection, abolished cell-associated transmission to unprotected T cells, and maintained protection through 45 days, post-infection (dpi) after a higher dose of HIV-1 infection. Finally, we describe here for the first time the assembly of all-in-one lentiviral vectors containing three and six gRNAs targeting tat and rev. A single-vector tat-targeting construct shows non-inferiority to the tat-targeting multi-vector in low-dose HIV-1 infection. We conclude that Cas9-induced, DNA repair-mediated mutations in tat are sufficiently deleterious and deplete HIV-1 fitness, and multiplexed disruption of tat further limits the possibility of an escape mutant arising, thus elevating the potential of CRISPR-Cas9 to achieve a long-term HIV-1 cure.

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Estudios in vitro de la capacidad mutagénica y potencialidad carcinogénica de sales metálicas e hidrocarburos aromáticos policíclicos por análisis a nivel citológico y molecular

10.35537/10915/4561 ◽

2004 ◽

Author(s):

◽

Silvana Andrea Mourón

Keyword(s):

Cáncer De Pulmón ◽

Exon 1

Los agentes carcinogénicos presentes en el ambiente son los principales agentes causales del desarrollo de la enfermedad neoplásica ya sea por la acción genotóxica directa (iniciador tumoral) o bien por la acción epigenética (promotor tumoral) que estimula la división celular y favorece la expresión del gen mutado antes, debido a la acción del iniciador. Por tanto, el estudio de la capacidad mutagénica y potencialidad carcinogénica de los contaminantes ambientales, podría brindar información para ayudar a dilucidar los mecanismos por los cuales estos compuestos ejercen su poder carcinogénico. Numerosos estudios han asociado la exposición de las sales de cadmio y de arsénico y de los hidrocarburos aromáticos policíclicos con el desarrollo de cáncer de pulmón. Sin embargo, los mecanismos mediante los cuales estas sustancias ejercen su poder carcinogénico no han sido elucidados cabalmente. A tal fin se empleó la línea de fibroblastos de pulmón humano MRC-5, y se evaluaron los efectos genotóxicos de las sales de cadmio (cloruro y sulfato de cadmio), las sales de arsénico (arsenito de sodio y ácido dimetilarsínico) y los hidrocarburos aromáticos policíclos (benzo[a]pireno y dibenzo[a,i]pireno). Se evaluó la capacidad de estos compuestos de inducir intercambios de cromátidas hermanas así como de rupturas de cadena simple de la molécula de ADN y /o la formación de aductos ADN-ADN o ADN-proteína e inducción de apoptosis mediante el empleo del ensayo cometa. Por otra parte, se evaluó la capacidad de inducción de mutaciones puntuales en el exón 1 del protooncogén K-ras y los exones 5 a 8 del gen supresor de tumores p53 mediante el empleo de la técnica de PCR-SSCP. Dado que las mutaciones puntuales en el codón 12 del protooncogén K-ras son las de mayor prevalencia e implicancia funcional del gen se evaluó su presencia mediante la técnica de PCR con enriquecimiento alélico.

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Proteomics-Based Characterization of the Effects of MMP14 on the Protein Content of Exosomes from Corneal Fibroblasts

Protein and Peptide Letters ◽

10.2174/0929866527666200408142827 ◽

2020 ◽

Vol 27 (10) ◽

pp. 979-988

Author(s):

Kyu-Yeon Han ◽

Jin-Hong Chang ◽

Dimitri T. Azar

Keyword(s):

Protein Content ◽

Catalytic Domain ◽

Protein Composition ◽

Proteomics Analysis ◽

Knock Out ◽

Corneal Fibroblast ◽

Flow Cytometric ◽

Corneal Fibroblasts ◽

Liquid Chromatography Tandem Mass

Background: Exosomes secreted by corneal fibroblasts contain matrix metalloproteinase (MMP) 14, which is known to influence pro-MMP2 accumulation on exosomes. Accordingly, we hypothesized that the enzymatic activity of MMP14 may alter the protein content of corneal fibroblast- secreted exosomes. Objective: The aim of this study was to investigate the effects of MMP14 on the composition and biological activity of corneal fibroblast-derived exosomes. Methods: Knock out of the catalytic domain (ΔExon4) of MMP14 in corneal fibroblasts was used to determine the effect of MMP14 expression on the characteristics of fibroblast-secreted exosomes. The amount of secreted proteins and their size distribution were measured using Nano Tracking Analysis. Proteins within exosomes from wild-type (WT) and ΔExon4-deficient fibroblasts were identified by liquid chromatography-tandem mass spectrometry (MS/MS) proteomics analysis. The proteolytic effects of MMP14 were evaluated in vitro via MS identification of eliminated proteins. The biological functions of MMP14-carrying exosomes were investigated via fusion to endothelial cells and flow cytometric assays. Results: Exosomes isolated from WT and ΔExon4-deficient fibroblasts exhibited similar size distributions and morphologies, although WT fibroblasts secreted a greater amount of exosomes. The protein content, however, was higher in ΔExon4-deficient fibroblast-derived exosomes than in WT fibroblast-derived exosomes. Proteomics analysis revealed that WT-derived exosomes included proteins that regulated cell migration, and ΔExon4 fibroblast-derived exosomes contained additional proteins that were cleaved by MMP14. Conclusion: Our findings suggest that MMP14 expression influences the protein composition of exosomes secreted by corneal fibroblasts, and through those biological components, MMP14 in corneal fibroblasts derived-exosomes may regulate corneal angiogenesis.

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Nanovesicles Loaded with Origanum onites and Satureja thymbra Essential Oils and Their Activity against Food-Borne Pathogens and Spoilage Microorganisms

Molecules ◽

10.3390/molecules26082124 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2124

Author(s):

Giulia Vanti ◽

Ekaterina-Michaela Tomou ◽

Dejan Stojković ◽

Ana Ćirić ◽

Anna Rita Bilia ◽

...

Keyword(s):

Essential Oils ◽

Propylene Glycol ◽

Antimicrobial Properties ◽

Spherical Shape ◽

Food Borne Pathogens ◽

Food Borne ◽

Spoilage Microorganisms ◽

Satureja Thymbra ◽

Origanum Onites

Food poisoning is a common cause of illness and death in developing countries. Essential oils (EOs) could be effective and safe natural preservatives to prevent and control bacterial contamination of foods. However, their high sensitivity and strong flavor limit their application and biological effectiveness. The aim of this study was firstly the chemical analysis and the antimicrobial evaluation of the EOs of Origanum onites L. and Satureja thymbra L. obtained from Symi island (Greece), and, secondly, the formulation of propylene glycol-nanovesicles loaded with these EOs to improve their antimicrobial properties. The EOs were analyzed by GC-MS and their chemical contents are presented herein. Different nanovesicles were formulated with small average sizes, high homogeneity, and optimal ζ-potential. Microscopic observation confirmed their small and spherical shape. Antibacterial and antifungal activities of the formulated EOs were evaluated against food-borne pathogens and spoilage microorganisms compared to pure EOs. Propylene glycol-nanovesicles loaded with O. onites EO were found to be the most active formulation against all tested strains. Additionally, in vitro studies on the HaCaT cell line showed that nanovesicles encapsulated with EOs had no toxic effect. The present study revealed that both EOs can be used as alternative sanitizers and preservatives in the food industry, and that their formulation in nanovesicles can provide a suitable approach as food-grade delivery system.

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