Genome Editing of the Octoploid Fragaria x ananassa Using the CRISPR/Cas9 System

Due to its octoploid nature, gene functional analyses in the cultivated strawberry (Fragaria × ananassa) are commonly carried out via gene silencing using self-complementary “hairpin” double-stranded RNA (RNAi) constructs. However, this system is not always as efficient as expected. First, an efficient silencing of the target gene is not always achieved, and second, its effect might not be stable after several clonal propagations of the transgenic lines. Recently, genome editing is becoming an important biotechnological tool for gene functional analysis and crop improvement, in particular since the development of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system. To investigate the functionality of the CRISPR/Cas9 in strawberry, we designed two sgRNAs directed against two regions of the floral homeotic gene APETALA3 (AP3) in order to induce a deletion of around 200 nt. A vector containing both sgRNAs and Cas9 was used to transform leaf disks of F. × ananassa cv. Camarosa. Several independent stable transgenic lines displayed defects in stamen and fruit development, partially phenocopying that of the Arabidopsis ap3 mutants. Molecular analysis of the targeted AP3 locus indicated differences in gene editing among different transgenic lines and suggests mutations in all the possible AP3 alleles. Phenotypic analyses indicate that impaired fruit development might be caused by the lack of proper development of the anthers due to the CRISPR/Cas9 induced mutation in AP3. In summary, we show that the CRISPR/Cas9 system is a functional tool to perform genome editing in the octoploid F. × ananassa. We propose this system as an alternative to the traditional RNAi strategy to stably mutagenize a particular gene of interest for functional analyses in this species.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Unusual DNA binding modes for metal anticancer complexes

DNA is believed to be the primary target for many metal-based drugs. For example, platinum-based anticancer drugs can form specific lesions on DNA that induce apoptosis. New platinum drugs can be designed that have novel modes of interaction with DNA, such as the trinuclear platinum complex BBR3464. Also it is possible to design inert platinum(IV) pro-drugs which are non-toxic in the dark, but lethal when irradiated with certain wavelengths of light. This gives rise to novel DNA lesions which are not as readily repaired as those induced by cisplatin, and provides the basis for a new type of photoactivated chemotherapy. Finally, newly emerging ruthenium(II) organometallic complexes not only bind to DNA coordinatively, but also by H-bonding and hydrophobic interactions triggered by the introduction of extended arene rings into their versatile structures. Intriguingly osmium (the heavier congener of ruthenium) reacts differently with DNA but can also give rise to highly cytotoxic organometallic complexes

The Andes mountain range uplift as a vicariant event in the Pimeliinae (Coleoptera: Tenebrionidae) in southern South America

The Andes mountain range extends over 8500 km along the Pacific coast of South America. Its medium altitude is 3500 m, reaching more than 6000 m at different latitudes. The uplift of the Andes  split arid habitats creating very diverse ecosystems on both sides. The distribution of the Pimeliinae (Coleoptera: Tenebrionidae) south of parallel 15º South is analysed, where the Andean mountain range separates xeric habitats both eastward and westward. The genera of Pimeliinae show four distribution patterns: endemic taxa east of the Andes, endemic taxa west of the Andes, taxa widely distributed on both sides of the Andes, and taxa inhabiting high altitudes in the Andes. Known phylogenies of genera and species of Pimeliinae are examined in terms of the Andean mountain uplift creating a vicariant event. A biogeographical track exhibited by certain genera of Pimeliinae connecting central Chile and southern Argentina is examined.La vaste étendue des Andes couvre plus que 8500 km le long de la côte pacifique de l’Amérique du sud. L’altitude moyenne est de 3500 m mais elle dépasse plus que 6000 m avec des écosystemes très variés des deux côtés. La distribution des Pimeliinae (Coleoptera : Tenebrionidae) est examinée au sud du 15eme parallèlle où la formation des Andes a séparé des habitats désertiques à la fois vers l’est et vers l’ouest. Les genres de Pimeliinae montrent quatre modèles de distribution : espèces endémiques à l’ouest des Andes ; espèces endémiques à l’est des Andes ; espèces largement répandues les deux côtés des Andes et espèces habitant les hautes altitudes des Andes. La phylogénie connue de certaines espèces et genres est examinée pour événement vicariant à la suite de la formation des montagnes andines. Un chemin biogeographique démontré par quelques genres de Pimeliinae liant le Chile central et l’Argentine du sud est analysé.Fil: Flores, Gustavo Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Provincia de Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Universidad Nacional de Cuyo. Instituto Argentino de Investigaciones de las Zonas Áridas; ArgentinaFil: Pizarro Araya, Jaime. Universidad de La Serena; Chil

The Story of the Puerto Rican-Chicano Committee

The concept or idea for The Puerto Rican-Chicano Committee (PRCC) came from UB student Alberto O. Cappas, who also was the original founder of PODER, at the time known as the Puerto Rican Organization for Dignity, Elevation, and Responsibility

STRUCTURAL AND PHOTOPHYSICAL PROPERTIES OF A MONONUCLEAR Re(I) COMPLEX:[P,N-{(C6H5)2(C5H5N)P}Re(CO)3Br]

Indexación: Web of Science; ScieloWe have prepared a new monometallic rhenium(I) carbonyl, the complex [P,N-{(C6H5)2(C5H5N)P}Re(CO)3Br] by direct reaction of (Re(CO)3Br(THF))2 and the ligand (C6H5)2(C5H5N)P The structure in the complex shows the pyridinic-phosphine ligand in a chelating mode, occupying cis positions around the rhenium octahedral environment. The molecule displays an absorption band centered at 315 nm which has been assigned to a MLCT transition, and a luminescence quantum yield of 0.001.http://ref.scielo.org/ptnng