Recent advances in metabolic regulation and bioengineering of gibberellic acid biosynthesis in Fusarium fujikuroi.

The plant growth hormone gibberellic acid (GA3), as one of the representative secondary metabolites, is widely used in agriculture, horticulture and brewing industry. GA3 is detected in both plants and several fungi with the ability to stimulate plant growth. Currently, the main mode of industrial production of GA3 is depended on the microbial fermentation via long-period submerged fermentation using Fusarium fujikuroi as the only producing strain, qualified for its natural productivity. However, the demand of large-sale industrialization of GA3 was still restricted by the low productivity. The biosynthetic route of GA3 in F. fujikuroi is now well-defined. Furthermore, the multi-level regulation mechanisms involved in the whole network of GA3 production have also been gradually unveiled by the past two decades based on the identification and characterization of several global regulators and their mutual functions. Combined with the quick development of genetic manipulation techniques, the rational modification of producing strain F. fujikuroi development become practical for higher productivity achievement. Herein, we review the latest advances in the molecular regulation of GA3 biosynthesis in F. fujikuroi and conclude a comprehensive network involving nitrogen depression, global regulator, histone modification and G protein signaling pathway. Correspondingly, the bioengineering strategies covering conventional random mutation, genetic manipulating platform development, metabolic edition and fermentation optimization were also systematically proposed.

Severe neurometabolic phenotype in npc1 -/- zebrafish with a C-terminal mutation.

Niemann Pick disease type C (NPC) is an autosomal recessive neurodegenerative lysosomal disorder characterized by an accumulation of lipids in different organs. Clinical manifestations can start at any age and include hepatosplenomegaly, intellectual impairment, and cerebellar ataxia. NPC1 is the most common causal gene, with over 460 different mutations with heterogeneous pathological consequences. We generated a zebrafish NPC1 model by CRISPR/Cas9 carrying a homozygous mutation in exon 22, which encodes the end of the cysteine-rich luminal loop of the protein. This is the first zebrafish model with a mutation in this gene region, which is frequently involved in the human disease. We observed a high lethality in npc1 mutants, with all larvae dying before reaching the adult stage. Npc1 mutant larvae were smaller than wild type (wt) and their motor function was impaired. We observed vacuolar aggregations positive to cholesterol and sphingomyelin staining in the liver, intestine, renal tubules and cerebral gray matter of mutant larvae. RNAseq comparison between npc1 mutants and controls showed 284 differentially expressed genes, including genes with functions in neurodevelopment, lipid exchange and metabolism, muscle contraction, cytoskeleton, angiogenesis, and hematopoiesis. Lipidomic analysis revealed significant reduction of cholesteryl esters and increase of sphingomyelin in the mutants. Compared to previously available zebrafish models, our model seems to recapitulate better the early onset forms of the NPC disease. Thus, this new model of NPC will allow future research in the cellular and molecular causes/consequences of the disease and on the search for new treatments.

Transgenesis in parasitic helminths: a brief history and prospects for the future.

Helminth infections impact the health of hundreds of millions of persons globally and also cause important economic losses in livestock farming. Methodological limitations as well as the low attention given to the study of helminths have impacted biological research and, thus, the procurement of accurate diagnosis and effective treatments. Understanding the biology of helminths using genomic and proteomic approaches could contribute to advances in understanding host-helminth interactions and lead to new vaccines, drugs and diagnostics. Despite the significant advances in genomics in the last decade, the lack of methodological adaptation of current transgenesis techniques has hampered the progression of post-genomic research in helminthology. However, the application of new techniques, such as CRISPR, to the study of trematodes and nematodes has opened new avenues for genome editing-powered functional genomics for these pathogens. This review summarises the historical advances in functional genomics in parasitic helminths and highlights pending limitations that will need to be overcome to deploy transgenesis tools.

A nop56 Zebrafish Loss-of-Function Model Exhibits a Severe Neurodegenerative Phenotype.

NOP56 belongs to a C/D box small nucleolar ribonucleoprotein complex that is in charge of cleavage and modification of precursor ribosomal RNAs and assembly of the 60S ribosomal subunit. An intronic expansion in NOP56 gene causes Spinocerebellar Ataxia type 36, a typical late-onset autosomal dominant ataxia. Although vertebrate animal models were created for the intronic expansion, none was studied for the loss of function of NOP56. We studied a zebrafish loss-of-function model of the nop56 gene which shows 70% homology with the human gene. We observed a severe neurodegenerative phenotype in nop56 mutants, characterized mainly by absence of cerebellum, reduced numbers of spinal cord neurons, high levels of apoptosis in the central nervous system (CNS) and impaired movement, resulting in death before 7 days post-fertilization. Gene expression of genes related to C/D box complex, balance and CNS development was impaired in nop56 mutants. In our study, we characterized the first NOP56 loss-of-function vertebrate model, which is important to further understand the role of NOP56 in CNS function and development.

Zebrafish Models of Autosomal Recessive Ataxias.

Autosomal recessive ataxias are much less well studied than autosomal dominant ataxias and there are no clearly defined systems to classify them. Autosomal recessive ataxias, which are characterized by neuronal and multisystemic features, have significant overlapping symptoms with other complex multisystemic recessive disorders. The generation of animal models of neurodegenerative disorders increases our knowledge of their cellular and molecular mechanisms and helps in the search for new therapies. Among animal models, the zebrafish, which shares 70% of its genome with humans, offer the advantages of being small in size and demonstrating rapid development, making them optimal for high throughput drug and genetic screening. Furthermore, embryo and larval transparency allows to visualize cellular processes and central nervous system development in vivo. In this review, we discuss the contributions of zebrafish models to the study of autosomal recessive ataxias characteristic phenotypes, behavior, and gene function, in addition to commenting on possible treatments found in these models. Most of the zebrafish models generated to date recapitulate the main features of recessive ataxias.

Zebrafish Models of Autosomal Dominant Ataxias.

Hereditary dominant ataxias are a heterogeneous group of neurodegenerative conditions causing cerebellar dysfunction and characterized by progressive motor incoordination. Despite many efforts put into the study of these diseases, there are no effective treatments yet. Zebrafish models are widely used to characterize neuronal disorders due to its conserved vertebrate genetics that easily support genetic edition and their optic transparency that allows observing the intact CNS and its connections. In addition, its small size and external fertilization help to develop high throughput assays of candidate drugs. Here, we discuss the contributions of zebrafish models to the study of dominant ataxias defining phenotypes, genetic function, behavior and possible treatments. In addition, we review the zebrafish models created for X-linked repeat expansion diseases X-fragile/fragile-X tremor ataxia. Most of the models reviewed here presented neuronal damage and locomotor deficits. However, there is a generalized lack of zebrafish adult heterozygous models and there are no knock-in zebrafish models available for these diseases. The models created for dominant ataxias helped to elucidate gene function and mechanisms that cause neuronal damage. In the future, the application of new genetic edition techniques would help to develop more accurate zebrafish models of dominant ataxias.

El Derecho ante la técnica de edición genética CRISPR / O Direito ante a técnica da edição genética. CRISPR / The Law in front of the gene editing technique. CRISPR

Resumen: La técnica de edición genética CRISPR constituye uno de los avances recientes más extraordinarios en materia de biotecnología, de alcances insospechados. En ese contexto, resulta fundamental analizar sus repercusiones en el ámbito de los derechos humanos, toda vez que, así como puede propiciar avances extraordinarios para su mejor desarrollo, puede también generar nuevas amenazas.

Resumo: A técnica de edição genética CRISPR constitui um dos avanços recentes mais extraordinários em matéria de biotecnologia, de alcances insuspeitos. Neste contexto, é fundamental analisar suas repercussões no âmbito dos direitos humanos, toda vez que, assim como pode propiciar avanços extraordinários para seu melhor desenvolvimento, pode também gerar novas ameaças.

Abstract: The CRISPR genetic editing technique is one of the most extraordinary recent advances in biotechnology, whose scope is unsuspected. In this context, it is fundamental to analyze its repercussions in the field of the Human Rights, since as well as it can promote extraordinary advances for its better development, it can also generate new threats.

CRISPR/Cas9: reflexiones bioéticas sobre las modificacion es genómicas / CRISPR/Cas9: bioethical reflections on genome modifications

CRISPR/Cas9 puede ser considerado el descubrimiento biotecnológico del siglo. Sin embargo, las reflexiones sobre la aceptabilidad y factibilidad de producir cambios permanentes en el ADN de gametos y embriones, ha arrojado nueva luz sobre CRISPR. Por ejemplo al notar que las alteraciones en la línea germinal, pueden ser heredadas y por lo tanto observarse a lo largo de las generaciones. Aunque inicialmente estas transformaciones puedan ser deseables, más allá del impacto tecnológico, la tecnología CRISPR parece tener varias implicancias éticas para la sociedad. Estas pueden ser analizadas a partir de las distintas reacciones que ha despertado CRISPR en todo el mundo. Por ejemplo, en el pedido de suspensión total de la aplicación clínica (es decir, la prohibición de transferir en el útero un embrión previamente modificado con la tecnología CRISPR) hasta tanto no se hayan alcanzado y aprobado ciertos requisitos indispensables para la investigación clínica. En general, esta tecnología ha sido prematuramente caracterizada como "disruptiva". Este texto analizará las implicancias éticas, políticas, sociales, médicas y subjetivas a la luz del fenómeno social - no tecnológico - creado por CRISPR.

CRISPR/Cas9 can be considered the biotechnogical discovery of the century. However, reflections on the plausibility and feasibility of producing permanent changes in the DNA of gametes and embryos throw new light on CRISPR. As in modifications in the germinal line can be inherited and hence, observed throughout generations. While this might be desirable for some, besides the technological impact, CRISPR also seems to have an ethical impact on society. These ethical impacts can be observed in the diverse reactions to CRISPR from across the globe. For instance, a request for the complete suspension of clinical application (that is, the prohibition of implanting an embryo with CRISPR modifications in the uterus) till certain basic research requirements were met and approved. Broadly, this technology has been prematurely also characterized as "disruptive" by some. This paper will analyze these ethical, political, social, medical and subjective reactions in light of the social - not technological - phenomenon created by CRISPR.