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CRISPR is a revolutionary gene editing technology that has enabled scientists worldwide to explore the cell's genetic blueprint in an unprecedented easy way. In this chapter, we will briefly present the history behind the development of this innovative tool, how it emerged from a natural bacterial mechanism for antiviral defense, its key components (Cas9 endonuclease and single guide RNA), mode of action (DNA cleavage and repair via NHEJ or HDR), and versatility (acting on single- or double-stranded DNA or RNA) for diverse purposes beyond gene editing such as stochastic marking, digital encoding, high-fidelity SNP genotyping, programmed chromosome fission/fusion, gene mapping, nucleic acid detection, regulation of gene expression, DNA/RNA labeling or tracking, and more.

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Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on in vitro tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars.

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Drought is the most detrimental abiotic stress to sugarcane production. Nevertheless, transcriptomic analyses remain scarce for field-grown plants. Here we performed comparative transcriptional profiling of two contrasting sugarcane genotypes, 'IACSP97-7065' (drought-sensitive) and 'IACSP94-2094' (drought-tolerant) grown in a drought-prone environment. Physiological parameters and expression profiles were analyzed at 42 (May) and 117 (August) days after the last rainfall. The first sampling was done under mild drought (soil water potential of -60 kPa), while the second one was under severe drought (soil water potential of -75 kPa). Microarray analysis revealed a total of 622 differentially expressed genes in both sugarcane genotypes under mild and severe drought stress, uncovering about 250 exclusive transcripts to 'IACSP94-2094' involved in oxidoreductase activity, transcriptional regulation, metabolism of amino acids, and translation. Interestingly, the enhanced antioxidant system of 'IACSP94-2094' may protect photosystem II from oxidative damage, which partially ensures stable photochemical activity even after 117 days of water shortage. Moreover, the tolerant genotype shows a more extensive set of responsive transcription factors, promoting the fine-tuning of drought-related molecular pathways. These results help elucidate the intrinsic molecular mechanisms of a drought-tolerant sugarcane genotype to cope with ever-changing environments, including prolonged water deficit, and may be useful for plant breeding programs.

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MicroRNAs (miRNAs) are small non-coding RNA molecules able to post-transcriptionally regulate gene expression via base-pairing with partially complementary sequences of target transcripts. Prion diseases comprise a singular group of neurodegenerative conditions caused by endogenous, misfolded pathogenic (prion) proteins, associated with molecular aggregates. In humans, classical prion diseases include Creutzfeldt-Jakob disease, fatal familial insomnia, Gerstmann-Sträussler-Scheinker syndrome, and kuru. The aim of this review is to present the connections between miRNAs and prions, exploring how the interaction of both molecular actors may help understand the susceptibility, onset, progression, and pathological findings typical of such disorders, as well as the interface with some prion-like disorders, such as Alzheimer's. Additionally, due to the inter-regulation of prions and miRNAs in health and disease, potential biomarkers for non-invasive miRNA-based diagnostics, as well as possible miRNA-based therapies to restore the levels of deregulated miRNAs on prion diseases, are also discussed. Since a cure or effective treatment for prion disorders still pose challenges, miRNA-based therapies emerge as an interesting alternative strategy to tackle such defying medical conditions.

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It is possible to gain a deeper insight into the role of water in biology by using physicochemical variant molecules, such as deuterium oxide (D2 O); however, D2 O is toxic to multicellular organisms in high concentrations. By using a unique desiccation-rehydration process, we demonstrate that the anhydrobiotic nematode Panagrolaimus superbus is able to tolerate and proliferate in 99 % D2 O. Moreover, we analysed P. superbus' water-channel protein (aquaporin; AQP), which is associated with dehydration/rehydration, by comparing its primary structure and modelling its tertiary structure in silico. Our data evidence that P. superbus' AQP is an aquaglyceroporin, a class of water channel known to display a wider pore; this helps to explain the rapid and successful organismal influx of D2 O into this species. This is the first demonstration of an animal able to withstand high D2 O levels, thus paving a way for the investigation of the effects D2 O on higher levels of biological organization.

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Panagrolaimus superbus nematodes are able to tolerate desiccation by entering into a peculiar state of suspended animation known as anhydrobiosis. When desiccated, anhydrobiotic organisms are also able to tolerate other physical stresses, as high and low levels of temperature and pressure. Here, we decided to investigate the tolerance of desiccated P. superbus to an unprecedented double stress - hypoxia within 99.99% Gallium (Ga) metal cage. The authors observed that regardless of the external relative humidity, desiccated P. superbus tolerated 7 d confined within the metal cage, displaying no negative effects on its survival and population growth rates over 40 d. The results evidence that anhydrobiosis also renders nematodes tolerant to otherwise lethal concentrations of Ga, in an oxygen-poor environment; thus, expanding its polyextremotolerance profile.Panagrolaimus superbus nematodes are able to tolerate desiccation by entering into a peculiar state of suspended animation known as anhydrobiosis. When desiccated, anhydrobiotic organisms are also able to tolerate other physical stresses, as high and low levels of temperature and pressure. Here, we decided to investigate the tolerance of desiccated P. superbus to an unprecedented double stress ­ hypoxia within 99.99% Gallium (Ga) metal cage. The authors observed that regardless of the external relative humidity, desiccated P. superbus tolerated 7 d confined within the metal cage, displaying no negative effects on its survival and population growth rates over 40 d. The results evidence that anhydrobiosis also renders nematodes tolerant to otherwise lethal concentrations of Ga, in an oxygen-poor environment; thus, expanding its polyextremotolerance profile.

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RNA interference (RNAi) is a powerful gene silencing technology, widely used in analyses of reverse genetics, development of therapeutic strategies and generation of biotechnological products. Here we present a free software tool for the rational design of RNAi effectors, named siRNA and shRNA designer (SSD). SSD incorporates our previously developed software Strand Analysis to construct template DNAs amenable for the large scale production of mono-, bi- and trivalent multimeric shRNAs, via in vitro rolling circle transcription. We tested SSD by creating a trivalent multimeric shRNA against the vitellogenin gene of Apis mellifera. RT-qPCR analysis revealed that our molecule promoted a decrease in more than 50% of the target mRNA, in a dose-dependent manner, when compared to the control group. Thus, SSD software allows the easy design of multimeric shRNAs, for single or multiple simultaneous knockdowns, which is especially interesting for studies involving large amounts of double-stranded molecules.

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Abdominal tumors (AT) in children account for approximately 17% of all pediatric solid tumor cases, and frequently exhibit embryonal histological features that differentiate them from adult cancers. Current molecular approaches have greatly improved the understanding of the distinctive pathology of each tumor type and enabled the characterization of novel tumor biomarkers. As seen in abdominal adult tumors, microRNAs (miRNAs) have been increasingly implicated in either the initiation or progression of childhood cancer. Moreover, besides predicting patient prognosis, they represent valuable diagnostic tools that may also assist the surveillance of tumor behavior and treatment response, as well as the identification of the primary metastatic sites. Thus, the present study was undertaken to compile up-to-date information regarding the role of dysregulated miRNAs in the most common histological variants of AT, including neuroblastoma, nephroblastoma, hepatoblastoma, hepatocarcinoma, and adrenal tumors. Additionally, the clinical implications of dysregulated miRNAs as potential diagnostic tools or indicators of prognosis were evaluated.

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One of the most important laboratory animal species is the nematode Caenorhabditis elegans, which has been used in a range of research fields such as neurobiology, body development, and molecular biology. The scientific progress obtained by employing C. elegans as a model in these areas has encouraged its use in new fields. One of the new potential applications concerns the biological responses to hyperacceleration stress (g-force), but only a few studies have evaluated the response of multicellular organisms to extreme hypergravity conditions at the order of magnitude 105 x g, which is the theorized force experienced by rocks ejected from Mars (or similar planets). Therefore, we subjected the nematode C. elegans to 400,000 x g (equivalent to that force) and evaluated viability, general morphology, and behavior of C. elegans after exposure to this stress. The metabolic activity of this nematode in response to the gravitational spectrum of 50-400,000 x g was also evaluated by means of the MTT assay. Surprisingly, we found that this organism showed no decrease in viability, no changes in behavior and development, and no drastic metabolic depression after hyperacceleration. Thus, we demonstrated for the first time that this multicellular research model can withstand extremely high g-forces, which prompts the use of C. elegans as a new model for extreme hypergravity. Key Words: Caenorhabditis elegans-Hypergravity-Ultracentrifugation-Acceleration-Panspermia-Astrobiology. Astrobiology 18, 825-833.

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Abstract The molecular basis of anhydrobiosis, the state of suspended animation entered by some species during extreme desiccation, is still poorly understood despite a number of transcriptome and proteome studies. We therefore conducted functional screening by RNA interference (RNAi) for genes involved in anhydrobiosis in the holo-anhydrobiotic nematode Panagrolaimus superbus. A new method of survival analysis, based on staining, and proof-of-principle RNAi experiments confirmed a role for genes involved in oxidative stress tolerance, while a novel medium-scale RNAi workflow identified a further 40 anhydrobiosis-associated genes, including several involved in proteostasis, DNA repair and signal transduction pathways. This suggests that multiple genes contribute to anhydrobiosis in P. superbus.