SUMMARY
Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR‐associated proteins (Cas) have revolutionized genome editing in many organisms, including ...plants. Most CRISPR‐Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target‐specific and a screening process is usually required to identify those most effective. Plant virus‐derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus‐induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX‐based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus‐free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR‐Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.
Significance Statement
Plant virus‐derived vectors allow fast and efficient delivery of synthetic guide RNAs into adult plants for CRISPR‐Cas‐based genome editing. We engineered a Potato virus X vector for CRISPR‐Cas genome editing of solanaceous plants. This vector expresses unspaced arrays of synthetic guide RNAs and achieves multiplex editing in adult plant tissues in a few days. Virus‐free multiplex genome‐edited plants with biallelic mutations can be easily obtained from inoculated plants.
Inflammatory bowel diseases (ulcerative colitis; Crohn's disease) are debilitating relapsing inflammatory disorders affecting the gastrointestinal tract, with deleterious effect on quality of life, ...and increasing incidence and prevalence. Mucosal inflammation, due to altered microbiota, increased intestinal permeability and immune system dysfunction underlies the symptoms and may be caused in susceptible individuals by different factors (or a combination of them), including dietary habits and components. In this review we describe the influence of the Western diet, obesity, and different nutraceuticals/functional foods (bioactive peptides, phytochemicals, omega 3-polyunsaturated fatty acids, vitamin D, probiotics and prebiotics) on the course of IBD, and provide some hints that could be useful for nutritional guidance. Hopefully, research will soon offer enough reliable data to slow down the spread of the disease and to make diet a cornerstone in IBD therapy.
Flavonoids are plant bioactive compounds of great interest in nutrition and pharmacology, due to their remarkable properties as antioxidant, anti-inflammatory, antibacterial, antifungal and antitumor ...drugs. More than 5000 different flavonoids exist in nature, with a huge structural diversity and a plethora of interesting pharmacological properties. In this work, five flavonoids were tested for their potential use as antitumor drugs against three CRC cell lines (HCT116, HT-29 and T84). These cell lines represent three different stages of this tumor, one of which is metastatic. Xanthohumol showed the best antitumor activity on the three cancer cell lines, even better than that of the clinical drug 5-fluorouracil (5-FU), although no synergistic effect was observed in the combination therapy with this drug. On the other hand, apigenin and luteolin displayed slightly lower antitumor activities on these cancer cell lines but showed a synergistic effect in combination with 5-FU in the case of HTC116, which is of potential clinical interest. Furthermore, a literature review highlighted that these flavonoids show very interesting palliative effects on clinical symptoms such as diarrhea, mucositis, neuropathic pain and others often associated with the chemotherapy treatment of CRC. Flavonoids could provide a double effect for the combination treatment, potentiating the antitumor effect of 5-FU, and simultaneously, preventing important side effects of 5-FU chemotherapy.
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•Five plant flavonoids have been tested for their potential as antitumor drugs against human colorectal cancer (CRC) cell lines.•NApigenin and luteolin (and less so naringenin and eriodictyol) showed interesting antitumor activities in vitro.•Xanthohumol displayed the greatest antiproliferative activity, even higher than the clinically used drug 5-fluorouracil.•All these antitumor flavonoids also exert very interesting palliative/preventive effects on clinical symptoms associated to CRC treatment.
SUMMARY
Programmable transcriptional regulators based on CRISPR architecture are promising tools for the induction of plant gene expression. In plants, CRISPR gene activation is effective with ...respect to modulating development processes, such as the flowering time or customizing biochemical composition. The most widely used method for delivering CRISPR components into the plant is Agrobacterium tumefaciens‐mediated genetic transformation, either transient or stable. However, as a result of their versatility and their ability to move, virus‐derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular guide RNA (gRNA), which represents the variable component in CRISPR strategies. In the present study, we describe a Potato virus X‐derived vector that, upon agroinfection in Nicotiana benthamiana, serves as a vehicle for delivery of gRNAs, producing highly specific virus‐induced gene activation. The system works in combination with a N. benthamiana transgenic line carrying the remaining complementary CRISPR gene activation components, specifically the dCasEV2.1 cassette, which has been shown previously to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non‐invasive spraying method, we show that gRNA‐mediated activation programs move locally and systemically, generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this Potato virus X‐based virus‐induced gene reprogramming strategy results in program‐specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid‐enriched metabolite profiles.
Significance Statement
The virus‐derived systems for delivering CRISPR components to the plants have emerged as alternatives to the classical methods of plant transformation as a result of their versatility and their movement capacity. The presented PVX_VIGR approach provides an optimized CRISPR gene activation‐guide RNA delivery that expands the toolbox for reprogramming gene expression in plants. In addition, an easily scalable, non‐invasive spraying optimized method for PVX_VIGR strategy results in a specific modulation of the metabolic content in N. benthamiana.
Summary
CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti‐CRISPR proteins as tools to prevent CRISPR/Cas‐mediated gene editing and gene activation in plants has not ...been explored yet. This study describes the characterization of two anti‐CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site‐directed mutagenesis in leaves when transiently co‐expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a‐mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas‐based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose‐dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin‐regulated activation of a downstream reporter gene. The strong anti‐Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.
Mast cells are key actors in inflammatory reactions. Upon activation, they release histamine, heparin and nerve growth factor, among many other mediators that modulate immune response and neuron ...sensitization. One important feature of mast cells is that their population is usually increased in animal models and biopsies from patients with irritable bowel syndrome (IBS). Therefore, mast cells and mast cell mediators are regarded as key components in IBS pathophysiology. IBS is a common functional gastrointestinal disorder affecting the quality of life of up to 20% of the population worldwide. It is characterized by abdominal pain and altered bowel habits, with heterogeneous phenotypes ranging from constipation to diarrhea, with a mixed subtype and even an unclassified form. Nutrient intake is one of the triggering factors of IBS. In this respect, certain components of the daily food, such as fatty acids, amino acids or plant-derived substances like flavonoids, have been described to modulate mast cells' activity. In this review, we will focus on the effect of these molecules, either stimulatory or inhibitory, on mast cell degranulation, looking for a nutraceutical capable of decreasing IBS symptoms.
The recent emergence of tools based on the clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR‐associated (Cas) proteins have revolutionized targeted genome editing, thus ...holding great promise to both basic plant science and precision crop breeding. Conventional approaches for the delivery of editing components rely on transformation technologies or transient delivery to protoplasts, both of which are time‐consuming, laborious, and can raise legal concerns. Alternatively, plant RNA viruses can be used as transient delivery vectors of CRISPR–Cas reaction components, following the so‐called virus‐induced genome editing (VIGE). During the last years, researchers have been able to engineer viral vectors for the delivery of CRISPR guide RNAs and Cas nucleases. Considering that each viral vector is limited to its molecular biology properties and a specific host range, here we review recent advances for improving the VIGE toolbox with a special focus on strategies to achieve tissue‐culture‐free editing in plants. We also explore the utility of CRISPR–Cas technology to enhance biotic resistance with a special focus on plant virus diseases. This can be achieved by either targeting the viral genome or modifying essential host susceptibility genes that mediate in the infection process. Finally, we discuss the challenges and potential that VIGE holds in future breeding technologies.
Core Ideas
Viral vectors are useful tools for the transient delivery of guide RNAs and Cas nucleases into plant cells.
Virus‐mediated delivery of CRISPR–Cas reaction components avoids limitations associated to stable transformation.
Resistance to virus diseases can be achieved by CRISPR–Cas editing of viral genes or host susceptibility factors.
Coffee is one of the most popular beverages consumed worldwide. Roasted coffee is a complex mixture of thousands of bioactive compounds, and some of them have numerous potential health-promoting ...properties that have been extensively studied in the cardiovascular and central nervous systems, with relatively much less attention given to other body systems, such as the gastrointestinal tract and its particular connection with the brain, known as the brain-gut axis. This narrative review provides an overview of the effect of coffee brew; its by-products; and its components on the gastrointestinal mucosa (mainly involved in permeability, secretion, and proliferation), the neural and non-neural components of the gut wall responsible for its motor function, and the brain-gut axis. Despite in vitro, in vivo, and epidemiological studies having shown that coffee may exert multiple effects on the digestive tract, including antioxidant, anti-inflammatory, and antiproliferative effects on the mucosa, and pro-motility effects on the external muscle layers, much is still surprisingly unknown. Further studies are needed to understand the mechanisms of action of certain health-promoting properties of coffee on the gastrointestinal tract and to transfer this knowledge to the industry to develop functional foods to improve the gastrointestinal and brain-gut axis health.
ABSTRACT Virus-induced genome editing (VIGE) leverages viral vectors to deliver CRISPR-Cas components into plants for robust and flexible trait engineering. We describe here a VIGE approach applying ...an RNA viral vector based on potato virus X (PVX) for genome editing of tomato, a mayor horticultural crop. Viral delivery of single-guide RNA into Cas9-expressing lines resulted in efficient somatic editing with indel frequencies up to 58%. By proof-of-concept VIGE of PHYTOENE DESATURASE (PDS) and plant regeneration from edited somatic tissue, we recovered loss-of-function pds mutant progeny displaying an albino phenotype. VIGE of STAYGREEN 1 (SGR1), a gene involved in fruit color variation, generated sgr1 mutant lines with recolored red-brown fruits and high chlorophyll levels. The obtained editing events were heritable, overall confirming the successful breeding of fruit color. Altogether, our VIGE approach offers great potential for accelerated functional genomics of tomato variation, as well as for precision breeding of novel tomato traits.
Abstract This study aims to investigate whether the egg white hydrolysate (EWH) acts on the neuropathic disorders associated with long-term Mercury (Hg) exposure in rats. 8- week-old male Wistar rats ...were treated for 60 days with: a) Control - saline solution ( i.m. ); b) Mercury - HgCl2 (1st dose 4.6 μg/kg, subsequent doses 0.07 μg/kg/day, i.m. ); c) Hydrolysate - EWH (1 g/kg/day, gavage); d) Mercury and Hydrolysate. Mechanical allodynia was assessed using Von Frey Hairs test; heat hyperalgesia by the plantar test; catalepsy by a modification of the “ring test” and spontaneous locomotor activity by a photocell activity chambers. Analyses were performed at 0, 30 and 60 days of treatment. Brain and plasma MDA, plasma NPSH and TNF-α determination and skin immunohistochemistry were performed at 60 days. Hg induced a reduction in mechanical sensitivity threshold at 30 and 60 days and in thermal sensitivity threshold at 60 days. At the end of treatment catalepsy was developed, but there was not significant alteration in spontaneous locomotor activity. Hg also increased brain and plasma MDA, plasma NPSH and TNF- α levels and the number of Merkel cell–neurite complex in the skin. EWH prevented the development of mechanical allodynia, thermal hyperalgesia and catalepsy induced by Hg and the increase in MDA concentration in brain and plasma and in the number of Merkel cell–neurite complex in the skin. In conclusion, EWH promotes neuroprotection against the toxic effects caused by Hg, demonstrating a beneficial therapeutic potential.
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