PhD Discussions Sessions: Andrés Marco and Davia Prischich

An iCRISPR Platform for Human Functional Genetics in Human Pluripotent Stem Cells (hPSCs)

Self-renewal and pluripotency are the two major functional properties defining human pluripotent stem cells (hPSCs), allowing them to be cultured indefinitely in a dish and maintaining their capacity to differentiate to virtually any human cell lineage, tissue or organ. Hence, they represent an ideal model for studying cellular and multicellular behaviors in both physiological and pathological conditions.

Up until recently, the genome of hPSCs was difficult to manipulate, limiting considerably their use for functional genetics. To solve this problem, here we present the results of our ongoing effort to engineer an iCRISPR platform for highly efficient genome engineering in hPSCs. We have targeted the safe harbor AAVS1 locus using an inducible Cas9 editing vector (iC2), an inducible dCas9 activator (iCa) or an inducible dCas9 repressor (iCr). iCRISPR allows inducible gene knockout, gene upregulation and gene repression.

 All together, these lines will greatly expand the repertoire of applications that can be addressed with hPSCs. Our final goal is to use the iCRISPR platform to dissect kidney development and disease in hPSC-derived kidney organoids.

Traffic Lights peptides to photocontrol clathrin-mediated endocytosis in yeasts

Clathrin-mediated endocytosis (CME) is crucial to all eukaryotic cells. It is implicated in a variety of cellular processes that range from nutrient uptake, signal transduction and regulation of the membrane components including surface proteins. The functioning of this transient machinery requires a complex network of proteins that cannot be untangled only by means of genetic modification and immunological depletion. In this sense, photopharmacology provides a powerful aid by complementing the selectivity of drugs with the remote and reversible control offered by light.

Traffic Lights (TLs) peptides are cell-permeable, photoswitchable inhibitors specifically developed to target the main adaptor complex of the CME machinery. These peptides, named TL1 and TL2, have already proved capable of inhibiting CME in a light-regulated manner when tested in mammalian cells. Here we show that TL peptides retain their activity in yeast. After having confirmed photoregulation of CME events in this extremely versatile eukaryotic model system, we now aim to achieve in situ activation of the peptide so to directly address the role of endocytosis in cellular processes such as cytokinesis or cell migration.

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