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Among the New Plant Breeding Techniques (NPBTs), the CRISPR/Cas9 system represents a useful tool for target gene editing, improving the traits of the plants rapidly. This technology allows targeting one or more sequences simultaneously, as well as introducing new genetic variations by homology-directed recombination. However, the technology of CRISPR/Cas9 remains a challenge for some polyploid woody species, since all the different alleles for which the mutation is required must be simultaneously targeted. In this work we describe improved protocols adapting the CRISPR/Cas9 system to highbush blueberry ( Vaccinium corymbosum L.), using Agrobacterium -mediated transformation. As a proof of concept, we targeted the gene encoding for phytoene desaturase , whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Leaf explants of in vitro -cultured blueberry cv. Berkeley has been transformed with a CRISPR/Cas9 construct containing two guide RNAs (gRNA1 and gRNA2) targeting two conserved gene regions of pds and subsequently maintained on a selection medium enriched with kanamycin. After 4 weeks in culture on the selection medium, the kanamycin-resistant lines were isolated, and the genotyping of these lines through Sanger sequencing revealed successful gene editing. Some of mutant shoot lines included albino phenotypes, even if the editing efficiencies were quite low for both gRNAs, ranging between 2.1 and 9.6% for gRNA1 and 3.0 and 23.8 for gRNA2. Here we showed a very effective adventitious shoot regeneration protocol for the commercial cultivar of highbush blueberry “Berkeley”, and a further improvement in the use of CRISPR/Cas9 system in Vaccinium corymbosum L., opening the way to the breeding mediated by biotechnological approaches.

Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1 - 10 ) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes ( SmelPPO4, SmelPPO5 , and SmelPPO6) , which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5 , and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T 1 and T 2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

In the Medicago genus, triterpene saponins are a group of bioactive compounds extensively studied for their different biological and pharmaceutical properties. In this work, the CRISPR/Cas9-based approach with two single-site guide RNAs was used in Medicago truncatula (barrel medic) to knock-out the CYP93E2 and CYP72A61 genes, which are responsible for the biosynthesis of soyasapogenol B, the most abundant soyasapogenol in Medicago spp. No transgenic plants carrying mutations in the target CYP72A61 gene were recovered while fifty-two putative CYP93E2 mutant plant lines were obtained following Agrobacterium tumefaciens -mediated transformation. Among these, the fifty-one sequenced plant lines give an editing efficiency of 84%. Sequencing revealed that these lines had various mutation patterns at the target sites. Four T0 mutant plant lines were further selected and examined for their sapogenin content and plant growth performance under greenhouse conditions. The results showed that all tested CYP93E2 knock-out mutants did not produce soyasapogenols in the leaves, stems and roots, and diverted the metabolic flux toward the production of valuable hemolytic sapogenins. No adverse influence was observed on the plant morphological features of CYP93E2 mutants under greenhouse conditions. In addition, differential expression of saponin pathway genes was observed in CYP93E2 mutants in comparison to the control. Our results provide new and interesting insights into the application of CRISPR/Cas9 for metabolic engineering of high-value compounds of plant origin and will be useful to investigate the physiological functions of saponins in planta .

BackgroundArtificial intelligence (AI) has the potential to enhance patient safety in surgery, and all its aspects, including education and training, will derive considerable benefit from AI. In the present study, deep-learning models were used to predict the rates of proficiency acquisition in robot-assisted surgery (RAS), thereby providing surgical programs directors information on the levels of the innate ability of trainees to facilitate the implementation of flexible personalized training.Methods176 medical students, without prior experience with surgical simulators, were trained to reach proficiency in five tasks on a virtual simulator for RAS. Ensemble deep neural networks (DNN) models were developed and compared with other ensemble AI algorithms, i.e., random forests and gradient boosted regression trees (GBRT).ResultsDNN models achieved a higher accuracy than random forests and GBRT in predicting time to proficiency, 0.84 vs. 0.70 and 0.77, respectively (Peg board 2), 0.83 vs. 0.79 and 0.78 (Ring walk 2), 0.81 vs 0.81 and 0.80 (Match board 1), 0.79 vs. 0.75 and 0.71 (Ring and rail 2), and 0.87 vs. 0.86 and 0.84 (Thread the rings 2). Ensemble DNN models outperformed random forests and GBRT in predicting number of attempts to proficiency, with an accuracy of 0.87 vs. 0.86 and 0.83, respectively (Peg board 2), 0.89 vs. 0.88 and 0.89 (Ring walk 2), 0.91 vs. 0.89 and 0.89 (Match board 1), 0.89 vs. 0.87 and 0.83 (Ring and rail 2), and 0.96 vs. 0.94 and 0.94 (Thread the rings 2).ConclusionsEnsemble DNN models can identify at an early stage the acquisition rates of surgical technical proficiency of trainees and identify those struggling to reach the required expected proficiency level.

Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model in eggplant, which is pivotal in the transcriptional regulation of the anthocyanin biosynthetic pathway. Through a genome-wide approach performed on the recently released Eggplant Genome (cv. 67/3) previously identified, and reconfirmed by us, members belonging to the MBW complex (SmelANT1, SmelAN2, SmelJAF13, SmelAN1) were functionally characterized. Furthermore, a regulatory R3 MYB type repressor (SmelMYBL1), never reported before, was identified and characterized as well. Through a qPCR approach, we revealed specific transcriptional patterns of candidate genes in different plant tissue/organs at two stages of fruit development. Two strategies were adopted for investigating the interactions of bHLH partners (SmelAN1, SmelJAF13) with MYB counterparts (SmelANT1, SmelAN2 and SmelMYBL1): Yeast Two Hybrid (Y2H) and Bimolecular Fluorescent Complementation (BiFC) in A. thaliana mesophylls protoplast. Agro-infiltration experiments highlighted that N. benthamiana leaves transiently expressing SmelANT1 and SmelAN2 showed an anthocyanin-pigmented phenotype, while their co-expression with SmelMYBL1 prevented anthocyanin accumulation. Our results suggest that SmelMYBL1 may inhibits the MBW complex via the competition with MYB activators for bHLH binding site, although this hypothesis requires further elucidation.

DNA methylation through the activity of cytosine-5-methyltransferases (C5-MTases) and DNA demethylases plays important roles in genome protection as well as in regulating gene expression during plant development and plant response to environmental stresses. In this study, we report on a genome-wide identification of six C5-MTases (SmelMET1, SmelCMT2, SmelCMT3a, SmelCMT3b, SmelDRM2, SmelDRM3) and five demethylases (SmelDemethylase_1, SmelDemethylase_2, SmelDemethylase_3, SmelDemethylase_4, SmelDemethylase_5) in eggplant. Gene structural characteristics, chromosomal localization and phylogenetic analyses are also described. The transcript profiling of both C5-MTases and demethylases was assessed at three stages of fruit development in three eggplant commercial F1 hybrids: i.e. 'Clara', 'Nite Lady' and 'Bella Roma', representative of the eggplant berry phenotypic variation. The trend of activation of C5-MTases and demethylase genes varied in function of the stage of fruit development and was genotype dependent. The transcription pattern of C5MTAses and demethylases was also assessed in leaves of the F1 hybrid 'Nite Lady' subjected to salt and drought stresses. A marked up-regulation and down-regulation of some C5-MTases and demethylases was detected, while others did not vary in their expression profile. Our results suggest a role for both C5-MTases and demethylases during fruit development, as well as in response to abiotic stresses in eggplant, and provide a starting framework for supporting future epigenetic studies in the species.

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase ( pds ), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications

BackgroundThere is an increasing interest for a test assessing objectively the innate aptitude for surgery as a craft specialty to complement the current selection process of surgical residents. The aim of this study was to quantify the size of individuals with high, average, and low level of innate psychomotor skills among medical students.MethodsA volunteer sample of 155 medical students, without prior experience with surgical simulator, executed five tasks at a virtual simulator for robot-assisted surgery. They had to reach proficiency twice consecutively in each before moving to the next one. A weighting based on time and number of attempts needed to reach proficiency was assigned to each task.ResultsNine students (5.8%) out of 155 significantly outperformed all the others on median (i.q.r.) weighted time [44.7 (42.2–47.3) min vs. 98.5 (70.8–131.8) min, p < 0.001], and number of attempts to reach proficiency [14 (12–15) vs. 23 (19–32.75), p < 0.001). Seventeen students (11.0%) scored significantly much worse than the rest on median weighted time [202.2 (182.5–221.0) min vs. 84.3 (65.7–114.4) min, p < 0.001], and number of attempts [42 (40–48) vs. 22 (17.25–28), p < 0.001]. Low correlation between simulator scores and extracurricular activities, like videogames and musical instruments, was found.ConclusionsThe test successfully identified two groups straddling the large cohort with average innate aptitude for psychomotor skills: (i) innately gifted and (ii) with scarce level. Hence, exercises on a virtual simulator are a valid test of innate manual dexterity and can be considered to complement the selection process for a surgical training program, primarily to identify individuals with low innate aptitude for surgery and advise them to consider specialization in other (non-craft) medical specialties.

Globe artichoke capitula are susceptible to browning due to oxidation of phenols caused by the activity of polyphenol oxidases (PPOs), this reduces their suitability for fresh or processed uses. A genome-wide analysis of the globe artichoke PPO gene family was performed. Bioinformatics analyses identified eleven PPOs and their genomic and amino acidic features were annotated. Cis -acting element analysis identified a gene regulatory and functional profile associated to plant growth and development as well as stress response. For some PPOs, phylogenetic analyses revealed a structural and functional conservation with different Asteraceae PPOs, while the allelic variants of the eleven PPOs investigated across four globe artichoke varietal types identified several SNP/Indel variants, some of which having impact on gene translation. By RTqPCR were assessed the expression patterns of PPOs in plant tissues and in vitro calli characterized by different morphologies. Heterogeneous PPO expression profiles were observed and three of them ( PPO6 , 7 and 11) showed a significant increase of transcripts in capitula tissues after cutting. Analogously, the same three PPOs were significantly up-regulated in calli showing a brown phenotype due to oxidation of phenols. Our results lay the foundations for a future application of gene editing aimed at disabling the three PPOs putatively involved in capitula browning.