Biology subjects

Garcia-Carpintero, V.

Publications and source records attributed to Garcia-Carpintero, V..

6 recordsLinked to original sources

bioRxiv2022

Dually biofortified cisgenic tomatoes with increased flavonoids and branched-chain amino acids content

Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically-engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide-resistant version of an acetolactate synthase (mSlALS) gene expressed broadly, and a MYB12-like transcription factor (SlMYB12) expressed in a fruit-specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21-fold compared to wild type levels), Valine (9-fold), Isoleucine (3-fold), and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64-fold) and quercetin (45-fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.

bioengineering↗

bioRxiv2020

K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

K-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented. The protocol was accompanied by software developed to aid with primer set design. As the first examples, K-seq in species as diverse as tomato, dog and wheat was developed. K-seq provided genetic distances similar to those based on WGS in dogs. Experiments comparing K-seq and GBS in tomato showed similar genetic results, although K-seq had the advantage of finding more SNPs for the same number of Illumina reads. The technology reproducibility was tested with two independent runs of the tomato samples, and the correlation coefficient of the SNP coverages between samples was 0.8 and the genotype match was above 94%. K-seq also proved to be useful in polyploid species. The wheat samples generated specific markers for all subgenomes, and the SNPs generated from the diploid ancestors were located in the expected subgenome with accuracies greater than 80%. K-seq is an open, patent-unencumbered, easy-to-set-up, cost-effective and reliable technology ready to be used by any molecular biology laboratory without special equipment in many genetic studies.

genetics↗

bioRxiv2020

Edition of complex gene families in tobacco with GoldenBraid 4.0, a multipurpose web-based platform for plant genome engineering

CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy in polyploid crops. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, multiplex constructs comprising tandemly arrayed gRNAs are difficult to assemble, this hampering more widespread use. Here we present a comprehensive upgrade of the popular cloning platform GoldenBraid (GB), in which, on top of its classical multigene cloning software, we integrate new assembly tools for two-dimensions gRNA multiplexing with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the gRNA unspaced approaches, respectively. As functional validation, we show, among others, the assembly of up to 17 tandemly-arrayed gRNAs constructs against a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in tobacco. With these constructs we generated a collection of Cas9-free SPL mutants harboring up to 9 biallelic mutations in a single generation. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR activators and repressors using single and multiplexing gRNAs is also validated. With the incorporation of the new CRISPR tools and parts collection, GB4.0 turns an unprecedentedly comprehensive open platform for plant genetic engineering.

molecular biology↗

bioRxiv2018

Strong gene activation with genome-wide specificity using a new orthogonal CRISPR/Cas9-based Programmable Transcriptional Activator.

Synthetic Biology (SynBio) aims at rewiring plant metabolic and developmental programs with orthogonal regulatory circuits. This endeavour requires new molecular tools able to interact with endogenous factors in a potent yet at the same time highly specific manner. A promising new class of SynBio tools that could play this function are the synthetic transcriptional activators based on CRISPR/Cas9 architecture, which combine autonomous activation domains (ADs) capable of recruiting the cells transcription machinery, with the easily customizable DNA-binding activity of nuclease-inactivated Cas9 protein (dCas9), creating so-called Programmable Transcriptional Activators (PTAs). In search for optimized dCas9-PTAs we performed a combinatorial analysis with seven different ADs arranged in four different protein/RNA architectures. This analysis resulted in the selection of a new dCas9-PTA with improved features as compared with previously reported activators. The new synthetic riboprotein, named dCasEV2.1, combines EDLL and VPR ADs using a multiplexable mutated version (v2.1) of the previously described aptamer-containing guide RNA2.0. We show here that dCasEV2.1 is a strong and wide spectrum activator, displaying variable activation levels depending on the basal activity of the target promoter. Maximum activation rates reaching up to 10000 fold were observed when targeting the NbDFR gene. Most remarkably, RNAseq analysis of dCasEV2.1-transformed N. benthamiana leaves revealed that the topmost activation capacity of dCasEV2.1 on target genes is accompanied with strict genome-wide specificity, making dCasEV2.1 an attractive tool for rewiring plant metabolism and regulatory networks.

synthetic biology↗

bioRxiv2018

Long-term phenotypic effects following vitrified-thawed embryo transfer in a rabbit model

Since the first human was conceived through in vitro fertilisation in 1978, over 6.5 million babies have been born by assisted reproductive technologies (ARTs). Although most ART babies and children seem healthy, in recent years several studies have evidenced a potential impact of ARTs on long-term development and health. Herein, we have developed an animal model to determine whether vitrified embryo transfer procedure induces phenotypic changes over the growth performance and in the complementary transcriptomic and proteomic analyses at hepatic level. To this end, 2 populations were developed; vitrified embryos transferred to the surrogate mothers (VT) and naturally conceived animals (NC). After delivery, animals were weighed weekly from 1 to 20 weeks of age. In adulthood, animals were euthanized and organs were harvested and weighed. After that, liver tissue was used to identify changes in the transcriptomic and proteomic profile. At adulthood, VT group showed significant lower body, liver and heart weight. After functional analysis of RNA-Seq data, a subset of 96 differentially expressed transcripts in VT animal were related to alteration in zinc homeostasis, lipid metabolism, and hepatic immune pathways. After proteomic analysis, a subset of 76 differentially expressed proteins also revealed some disturbed metabolic pathways related with the lipid and glycan metabolism, and an impaired oxidative metabolism related to ATP synthesis in the mitochondria. Current findings suggest that progeny derived after transfer of vitrified embryos have long-term consequences on growth rate and vital organs weights in adulthood, correlated with molecular signatures at transcriptomic and proteomic level of hepatic tissue.

developmental biology↗

bioRxiv2017

Large scale gene duplication affected the European eel (Anguilla anguilla) after the 3R teleost duplication

Genomic scale duplication of genes generates raw genetic material, which may facilitate new adaptations for the organism. Previous studies on eels have reported specific gene duplications, however a species-specific large-scale gene duplication has never before been proposed. In this study, we have assembled a de novo European eel transcriptome and the data show more than a thousand gene duplications that happened, according to a 4dTv analysis, after the teleost specific 3R whole genome duplication (WGD). The European eel has a complex and peculiar life cycle, which involves extensive migration, drastic habitat changes and metamorphoses, all of which could have been facilitated by the genes derived from this large-scale gene duplication.\n\nOf the paralogs created, those with a lower genetic distance are mostly found in tandem repeats, indicating that they are young segmental duplications. The older eel paralogs showed a different pattern, with more extensive synteny suggesting that a Whole Genome Duplication (WGD) event may have happened in the eel lineage. Furthermore, an enrichment analysis of eel specific paralogs further revealed GO-terms typically enriched after a WGD. Thus, this study, to the best of our knowledge, is the first to present evidence indicating an Anguillidae family specific large-scale gene duplication, which may include a 4R WGD.

genomics↗