Project description:Survey of post pollination events in a sexually deceptive orchid (Ophrys fusca): a transcriptional approach Pollination through deception is a widespread phenomenon in angiosperm, and is extremely common in Orchidaceae family. One of the most striking pollination mechanism in orchids is known as sexual deception, in which flowers lure pollinators by foraging chemical (sex pheromones), visual (e.g. labellum colour and/or shape) and tactile (e.g. labellum pilosity) cues of the female insect pollinator. Ophrys has been used as a model genus to study sexual deception mechanism, mainly regarding chemical analysis in plant-insect association. Study was focused on Ophrys fusca, a species widely distributed in Mediterranean Basin. The main objective rely on Ophrys fusca gene expression study after pollination, through a transcriptional approach using cDNA microarrays. In order to evaluate pollination enhanced events, two different time points were selected: 2 days and 4 days after pollination. Ophrys fusca plants were sampled from a Portuguese natural occurring population. Plants were covered with a white and inert net, built specially for preventing pollinator’s visits in both pollinated and unpollinated flowers. Cross- pollination was performed manually with a sterile plastic stick. Five biological replicates (5 plants in each replicate) from each condition (pollinated and unpollinated) were collected in each time-point Flowers that demonstrate strict pollination regulation, as orchids, provide an excellent model system to unravel pollination- elicited mechanisms (i.e. petal senescence, pigmentation changes, ovary growth). Therefore, this study aims to contribute to the overall knowledge on orchid pollination biology, which is still lacking.
Project description:Generation of transgenic cell lines is limited by inefficient gene editing requiring genotypic screening of hundreds to thousands of colonies to isolate correctly gene-edited cells. Here, we describe a novel method called CRISPRa On-Target Editing Retrieval (CRaTER) that enriches for cells with on-target knock-in of a promoterless cDNA-fluorescent reporter transgene by transiently overexpressing the targeted endogenous genetic locus and sorting for fluorescent cells. We use CRaTER to enrich for rare cells with heterozygous, biallelic-editing of the endogenous, transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSC), resulting in a 9-fold enrichment compared to antibiotics selection alone. We leveraged CRaTER to enrich for heterozygous knock-in of a library of single nucleotide variants (SNV) in MYH7, a gene encoding for sarcomeric MHC-β wherein autosomal dominant missense mutations cause cardiac and skeletal myopathies. CRaTER enabled 90% enrichment of heterozygous, biallelically-edited hiPSCs – a 38.6-fold enrichment compared to antibiotics selection alone – to generate 113 SNVs comprising 78 missense variants in MYH7. hiPSCs that have undergone CRaTER enrichment can differentiate to cardiomyocytes and exhibit expected localization and expression of MHC-β fusion proteins. Together, CRaTER substantially reduces the screening required for isolation of gene-edited cells, enabling the generation of transgenic cell lines at unprecedented scale.