scholarly journals CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0261331
Author(s):  
Laurie J. Mitchell ◽  
Valerio Tettamanti ◽  
Justin S. Rhodes ◽  
N. Justin Marshall ◽  
Karen L. Cheney ◽  
...  
Keyword(s):  
Gene Editing ◽  
Mutant Gene ◽  
Reef Fishes ◽  
Loss Of Function ◽  
Social Hierarchies ◽  
Sequential Hermaphroditism ◽  
Starting Point ◽  
Pattern Development ◽  
Encoding Gene ◽  
Biallelic Mutations

Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of zygotes was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR/Cas9 in A. ocellaris, as a platform for studying gene function in anemonefishes and other reef fishes.

scholarly journals CRISPR/Cas9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutants

2020 ◽  
Author(s):  
Laurie J. Mitchell ◽  
Valerio Tettamanti ◽  
Justin N. Marshall ◽  
Karen L. Cheney ◽  
Fabio Cortesi
Keyword(s):  
Gene Editing ◽  
Experimental Studies ◽  
Mutant Gene ◽  
Reef Fishes ◽  
Loss Of Function ◽  
Cell Stage ◽  
Social Hierarchies ◽  
Sequential Hermaphroditism ◽  
The One ◽  
Biallelic Mutations

ABSTRACTGenomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of eggs at the one-cell stage was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 50% of injected eggs. Further analysis of the subcloned mutant gene sequences revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in G0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful resource for future experimental studies that aim to elucidate gene function in anemonefishes and reef fishes in general.

Death Becomes Him: The Hypervisibility of Martyrdom and Invisibility of the Wounded in the Iconography of Lebanese Militarised Masculinities1

2020 ◽  
pp. 121-147
Author(s):  
Henri Myrttinen
Keyword(s):  
Civil Wars ◽  
Social Hierarchies ◽  
Starting Point ◽  
Visual Landscape ◽  
The Dead ◽  
Political Economic

The visual landscape of Lebanon both mirrors and reasserts the country’s complex socio-political, economic and gendered order. Using public memorialisations of the dead in the Lebanese and Syrian Civil Wars as a starting point, the chapter analyses how these reflect Lebanese realities and imaginaries, and how particular militarised masculinities are constructed through them. The chapter then contrasts these visualisations with the invisibilisation of conflict-related disabilities and the war-wounded and what these mean for the reproduction of gendered and other social hierarchies.

scholarly journals AHK3-Mediated Cytokinin Signaling Is Required for the Delayed Leaf Senescence Induced by SSPP

2019 ◽  
Vol 20 (8) ◽  
pp. 2043
Author(s):  
Yanan Wang ◽  
Xiyu Zhang ◽  
Yanjiao Cui ◽  
Lei Li ◽  
Dan Wang ◽  
...  
Keyword(s):  
Leaf Senescence ◽  
Protein Phosphatase ◽  
Developmental Process ◽  
Loss Of Function ◽  
Cytokinin Signaling ◽  
Phenotypic Analysis ◽  
Exogenous Cytokinin ◽  
Cytokinin Treatment ◽  
Encoding Gene ◽  
Multiple Signaling

Leaf senescence is a highly-programmed developmental process regulated by an array of multiple signaling pathways. Our group previously reported that overexpression of the protein phosphatase-encoding gene SSPP led to delayed leaf senescence and significantly enhanced cytokinin responses. However, it is still unclear how the delayed leaf senescence phenotype is associated with the enhanced cytokinin responses. In this study, we introduced a cytokinin receptor AHK3 knockout into the 35S:SSPP background. The phenotypic analysis of double mutant revealed that AHK3 loss-of-function reversed the delayed leaf senescence induced by SSPP. Moreover, we found the hypersensitivity of 35S:SSPP to exogenous cytokinin treatment disappeared due to the introduction of AHK3 knockout. Collectively, our results demonstrated that AHK3-mediated cytokinin signaling is required for the delayed leaf senescence caused by SSPP overexpression and the detailed mechanism remains to be further elucidated.

scholarly journals Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants

2020 ◽  
Vol 10 (6) ◽  
pp. 2033-2042 ◽  
Author(s):  
Renyu Li ◽  
Charles Vavrik ◽  
Cristian H. Danna
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Function ◽  
Good Predictor ◽  
Gene Editing ◽  
Selection Strategy ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Editing Event ◽  
Endogenous Gene ◽  
Non Coding Rnas

CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprogramming of Cas9 specificity using customizable small non-coding RNAs, and to the possibility of editing several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource-intensive. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxy genes. We found that each proxy predicted the occurrence of mutations in each surrogate gene with efficiencies ranging from 68 to 100%. The selection strategy laid out here provides a framework to facilitate the identification of multiplex edited plants, thus aiding in the study of gene function when functional redundancy hinders the effort to define gene-function-phenotype links.

scholarly journals Gene editing of the wheat homologs of TONNEAU 1‐recruiting motif encoding gene affects grain shape and weight in wheat

2019 ◽  
Vol 100 (2) ◽  
pp. 251-264 ◽  
Author(s):  
Wei Wang ◽  
Qianli Pan ◽  
Bin Tian ◽  
Fei He ◽  
Yueying Chen ◽  
...  
Keyword(s):  
Gene Editing ◽  
Grain Shape ◽  
Encoding Gene

scholarly journals Immunodeficient Rabbit Models: History, Current Status and Future Perspectives

2020 ◽  
Vol 10 (20) ◽  
pp. 7369
Author(s):  
Jun Song ◽  
Brooke Pallas ◽  
Dongshan Yang ◽  
Jifeng Zhang ◽  
Yash Agarwal ◽  
...  
Keyword(s):  
Gene Editing ◽  
Interleukin 2 ◽  
Current Status ◽  
Loss Of Function ◽  
Future Perspectives ◽  
Transcription Activator ◽  
Immune Genes ◽  
Forkhead Box ◽  
Recombination Activating Gene ◽  
Model Family

Production of immunodeficient (ID) models in non-murine animal species had been extremely challenging until the advent of gene-editing tools: first zinc finger nuclease (ZFN), then transcription activator-like effector nuclease (TALEN), and most recently clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR)/Cas9. We and others used those gene-editing tools to develop ID rabbits carrying a loss of function mutation in essential immune genes, such as forkhead box protein N1 (FOXN1), recombination activating gene 1/2 (RAG1/2), and interleukin 2 receptor subunit gamma (IL2RG). Like their mouse counterparts, ID rabbits have profound defects in their immune system and are prone to bacterial and pneumocystis infections without prophylactic antibiotics. In addition to their use as preclinical models for primary immunodeficient diseases, ID rabbits are expected to contribute significantly to regenerative medicine and cancer research, where they serve as recipients for allo- and xeno-grafts, with notable advantages over mouse models, including a longer lifespan and a much larger body size. Here we provide a concise review of the history and current status of the development of ID rabbits, as well as future perspectives of this new member in the animal model family.

scholarly journals Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis

2019 ◽  
Vol 78 (5) ◽  
pp. 676-682 ◽  
Author(s):  
Lan Zhao ◽  
Jian Huang ◽  
Yunshan Fan ◽  
Jun Li ◽  
Tianming You ◽  
...  
Keyword(s):  
Structural Damage ◽  
Drug Targets ◽  
Gene Editing ◽  
Joint Damage ◽  
Loss Of Function ◽  
Adeno Associated Virus ◽  
Structural Deterioration ◽  
Efficient Gene ◽  
Genes Encoding ◽  
Surgically Induced

ObjectivesOsteoarthritis (OA) is a painful and debilitating disease and it is associated with aberrant upregulation of multiple factors, including matrix metalloproteinase 13 (MMP13), interleukin-1β (IL-1β) and nerve growth factor (NGF). In this study, we aimed to use the CRISPR/Cas9 technology, a highly efficient gene-editing tool, to study whether the ablation of OA-associated genes has OA-modifying effects.MethodsWe performed intra-articular injection of adeno-associated virus, which expressed CRISPR/Cas9 components to target each of the genes encoding MMP13, IL-1β and NGF, in a surgically induced OA mouse model. We also tested triple ablations of NGF, MMP13 and IL-1β.ResultsLoss-of-function of NGF palliates pain but worsens joint damage in the surgically induced OA model. Ablation of MMP13 or IL-1β reduces the expression of cartilage-degrading enzymes and attenuates structural deterioration. Targeting both MMP13 and IL-1β significantly mitigates the adverse effects of NGF blockade on the joints.ConclusionsCRISPR-mediated ablation of NGF alleviates OA pain, and deletion of MMP13-1β or IL-1β attenuates structural damage in a post-traumatic OA model. Multiplex ablations of NGF, MMP13 and IL-1β provide benefits on both pain management and joint structure maintenance. Our results suggest that CRISPR-based gene editing is useful for the identification of promising drug targets and the development of feasible therapeutic strategies for OA treatment.

scholarly journals Framing Sálvame: Public debates on taste, quality and television in Spain

2018 ◽  
Vol 43 (2) ◽  
pp. 209-233 ◽  
Author(s):  
Reinald Besalú ◽  
Mercè Oliva ◽  
Óliver Pérez-Latorre
Keyword(s):  
Child Protection ◽  
Cultural Practices ◽  
Legal Issue ◽  
Regulatory Body ◽  
Social Hierarchies ◽  
Social Actors ◽  
Starting Point ◽  
The Social ◽  
Celebrity Gossip ◽  
Working Class Women

Abstract The main aim of this article is to analyze the social circulation of discourses on non-hegemonic cultural practices, in particular, on what is called “trash TV”, and how they are connected to struggles over cultural and social hierarchies. To do so, it takes a specific event as starting point: the injunction that the CNMC (the Spanish broadcasting regulatory body) filed against Mediaset (a commercial TV operator) to adjust the contents of Sálvame Diario (a celebrity gossip program frequently associated with “trash TV”) to the requirements of what is known as the “child protection time slot”. This paper uses constructionist framing to analyze how this event was discussed by different social actors. Our analysis shows that while the CNMC and the press painted the conflict as a legal issue, Sálvame and social media users focused their discussion on the social acceptability of celebrity gossip media and their viewers (specifically working-class women).

scholarly journals A gene-editing/complementation strategy for tissue-specific lignin reduction while preserving biomass yield

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hasi Yu ◽  
Chang Liu ◽  
Richard A. Dixon
Keyword(s):  
Gene Editing ◽  
Biomass Yield ◽  
Lignin Content ◽  
Open Reading Frame ◽  
General Strategy ◽  
Loss Of Function ◽  
Tissue Specific ◽  
Knock Out ◽  
Reading Frame ◽  
Endogenous Gene

Abstract Background Lignification of secondary cell walls is a major factor conferring recalcitrance of lignocellulosic biomass to deconstruction for fuels and chemicals. Genetic modification can reduce lignin content and enhance saccharification efficiency, but usually at the cost of moderate-to-severe growth penalties. We have developed a method, using a single DNA construct that uses CRISPR–Cas9 gene editing to knock-out expression of an endogenous gene of lignin monomer biosynthesis while at the same time expressing a modified version of the gene’s open reading frame that escapes cutting by the Cas9 system and complements the introduced mutation in a tissue-specific manner. Results Expressing the complementing open reading frame in vessels allows for the regeneration of Arabidopsis plants with reduced lignin, wild-type biomass yield, and up to fourfold enhancement of cell wall sugar yield per plant. The above phenotypes are seen in both homozygous and bi-allelic heterozygous T1 lines, and are stable over at least four generations. Conclusions The method provides a rapid approach for generating reduced lignin trees or crops with one single transformation event, and, paired with a range of tissue-specific promoters, provides a general strategy for optimizing loss-of-function traits that are associated with growth penalties. This method should be applicable to any plant species in which transformation and gene editing are feasible and validated vessel-specific promoters are available.

scholarly journals Highly efficient CRISPR gene editing in yeast enabled by double selection

2018 ◽  
Author(s):  
Philippe C Després ◽  
Alexandre K Dubé ◽  
Lou Nielly-Thibault ◽  
Nozomu Yachie ◽  
Christian R Landry
Keyword(s):  
High Frequency ◽  
Large Scale ◽  
Gene Editing ◽  
Chromosomal Translocations ◽  
Selection Strategy ◽  
Loss Of Function ◽  
Base Editing ◽  
Fitness Effects ◽  
Genetics And Genomics ◽  
Selection For

AbstractCRISPR-Cas9 loss of function (LOF) and base editing screens are powerful tools in genetics and genomics. Yeast is one of the main models in genetics and genomics, yet large-scale approaches remain to be developed in this species because of low mutagenesis rates without donor DNA. We developed a double selection strategy based on co-selection that increases LOF mutation rates, both for CRISPR-Cas9 and the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection for both approaches. Using modeling, we show that this improvement provides the required increased in detection power to measure the fitness effects of thousands of mutations in typical yeast pooled screens. We also show that multiplex genome editing with Cas9 causes programmable chromosomal translocations at high frequency, suggesting that multiplex editing should be performed with caution and that base-editors could be preferable tools for LOF screens.

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