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

CRISPR/Cas9-mediated editing of the TM6 MADS-box gene in the octoploid strawberry (Fragaria × ananassa)

The B-class of MADS-box transcription factors has been studied in many plant species, but remain uncharacterized in Rosaceae. APETALA3 (AP3) is a member of this family, controlling petal and stamen identities in Arabidopsis. In this study, we have identified two members of the AP3 linage in Fragaria × ananassa: FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. CRISPR/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA. In this work, we have used this genome editing system for the first time in an octoploid species in order to study the function of TM6 in strawberry flower development.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Targeted mutagenesis of FaTM6 in the Octoploid Strawberry (Fragaria x ananassa) using the CRISPR/Cas9 System

The B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in the Rosaceae family. APETALA3 (AP3), a member of this class, controls the identity of petals and stamens in Arabidopsis thaliana. In this work, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria × ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA. However, whether it can be efficiently used in an octoploid species such as F. × ananassa is not known. In our study, we report the application of the CRISPR/Cas9 in F. × ananassa to characterize the function of FaTM6 in flower development. An exhaustive analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a high efficiency genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in petal and especially in anther development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene functional analysis in an octoploid species such as F. × ananassa, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

CRISPR/Cas9-mediated editing of the TM6 MADS-box gene in the octoploid strawberry (Fragaria x ananassa)

El contenido del poster presentado está desarrollado en: doi:10.1093/jxb/ery400 y Publicado por Oxford University pressThe B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in the Rosaceae family. APETALA3 (AP3), a member of this class, controls the identity of petals and stamens in Arabidopsis thaliana. In this work, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria × ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA. However, whether it can be efficiently used in an octoploid species such as F. × ananassa is not known. In our study, we report the application of the CRISPR/Cas9 in F. × ananassa to characterize the function of FaTM6 in flower development. An exhaustive analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a high efficiency genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in petal and especially in anther development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene functional analysis in an octoploid species such as F. × ananassa, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Identification and functional characterization of transcription factors involved in flower development and fruit ripening in Fragaria x ananassa

Fecha de lectura de Tesis Doctoral: 22 de noviembre 2019Esta tesis doctoral está compuesta por tres capítulos, dos de los cuales han sido publicados: 1) Una revisión bibliográfica sobre el uso de las herramientas de edición génica para modular la maduración: Martín-Pizarro, C. and Posé, D (2018); Frontiers in Plant Science, 9(1415):1-8). 2) Un trabajo de investigación: Martín Pizarro, C. et al (2019); Journal of Experimental Botany, 3:885-895). El objetivo general de esta tesis ha sido la identificación y caracterización de factores de transcripción implicados tanto en el desarrollo de la flor de fresa, como en la regulación del proceso de maduración del fruto. Para el primer objetivo, se generaron líneas mutantes para un gen homeótico, TM6, en Fragaria x ananassa usando la metodología CRISPR/Cas9 por primera vez en esta especie. Para ello se puso a punto este sistema de edición génica en esta especie tan compleja (genoma octoploide y no secuenciado), y se obtuvieron plantas con un defecto en el desarrollo de pétalos y estambres, lo cual mostró que TM6 juega un papel fundamental para el correcto desarrollo de estos órganos. Para el segundo objetivo (no publicado hasta la fecha) se identificó un gen, denominado FaRIF, homólogo al gen NOR de tomate, el cual es clave para la regulación de la maduración. En nuestro trabajo estudiamos los patrones de expresión de este gen y generamos líneas transgénicas de silenciamiento y sobreexpresión, las cuales mostraron un claro retraso y aceleración de la maduración respectivamente. Se llevaron a cabo caracterizaciones fisiológicas y moleculares de dichas líneas, en especial estudios transcriptómicos y metabolómicos de líneas de silenciamiento. Estos análisis mostraron que FaRIF está implicado en la regulación de genes implicados en distintos aspectos de la maduración, como la acumulación de antocianinas, azúcares, ácidos orgánicos, lignina, etc. y el metabolismo de la pared celular

Functional analysis of TM6 MADS-Box gene in the octoploid strawberry by CRISPR/Cas9 directed mutagenesis

The B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in Rosaceae. APETALA3 (AP3), a member of this class, controls petal and stamen identities in Arabidopsis. In this study, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria x ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using CRISPR/Cas9 system is becoming a robust tool for targeted and stable DNA mutagenesis. However, whether it can be efficiently used in an octoploid species such as F. x ananassa is not yet established. Here we report the application of CRISPR/Cas9 to characterize the function of FaTM6 in strawberry flower development. An analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a highly efficient genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in anther development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene functional analysis in F. x ananassa as an octoploid species, and they offer new opportunities for engineering strawberry to improve traits of interest in breeding programs.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Study of Transcriptional Regulatory Network Controlling Strawberry Fruit Ripening and Quality

Ponencia invitadaRipening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project, we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. Also, we are using a collection of around two hundred wild strawberry (Fragaria vesca) accessions to identify loci involved in important traits such as aroma, size or resistance to pathogens. Finally, we are implementing the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect it might open opportunities for engineering this species to improve traits of economic importance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Photoactivation of trans diamine platinum complexes in aqueous solution and effect on reactivity towards nucleotides

We show that UVA irradiation (365 nm) of the Pt-IV complex trans,trans,trans-[(PtCl2)-Cl-IV(OH)(2)(dimethylamine) (isopropylamine)] (1), induces reduction to Pt-II photoproducts. For the mixed amine Pt-II complex, trans[(PtCl2)-Cl-II(isopropylamine)(methylamine)] (2), irradiation at 365 nm increases the rate and extent of hydrolysis, triggering the formation of diaqua species. Additionally, irradiation increases the extent of reaction of complex 2 with guanosine-5'-monophosphate and affords mainly the bis-adduct, while reactions with adenosine-5'-monophosphate and cytidine-5'-monophosphate give rise only to mono-nucleotide adducts. Density Functional Theory calculations have been used to obtain insights into the electronic structure of complexes 1 and 2, and their photophysical and photochemical properties. UVA-irradiation can contribute to enhanced cytotoxic effects of diamine platinum drugs with trans geometry

Functional Analysis of TM6 MADS-box gene in the Octoploid Strawberry by CRISPR/Cas9 directed mutagenesis

The B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in the Rosaceae family. APETALA3 (AP3), a member of this class, controls the identity of petals and stamens in Arabidopsis thaliana. In this work, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria x ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA. However, whether it can be efficiently used in an octoploid species such as F. ananassa is not known. Here we report for the first time the application of CRISPR/Cas9 in F. x ananassa to characterize the function of FaTM6 in flower development. An exhaustive analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a high efficiency genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in petal and especially in anther development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene functional analysis in an octoploid species such as F. x ananassa, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs