GEARS: A TOOLKIT FOR VISUALIZING AND MANIPULATING ENDOGENOUS PROTEIN FUNCTION скачать в хорошем качестве

GEARS: A TOOLKIT FOR VISUALIZING AND MANIPULATING ENDOGENOUS PROTEIN FUNCTION

Talk by Caroline Hoppe, Yale University, New Haven, USA of America at the 23rd HFSP Awardees Meeting (17-19 June 2024, Washington DC, USA) Abstract To probe endogenous protein localization and function in vivo, we have developed GEARs: Genetically Encoded Affinity Reagents; a collection of short epitope-based tags and their cognate GEAR binders, which can 1) measure protein localization and abundance and 2) perturb protein function in vivo. First, after validating the functionality of these reagents using exogenous reporters, we generated GEAR tagged alleles with a novel, rapid CRISPR/Cas9 based protein tagging pipeline in zebrafish. Second, we demonstrate that these tags can be used in conjunction with targeted protein degradation in zebrafish, mice and human cell culture. Next, we used GEARs to examine the native behavior of the pioneer transcription factor Nanog, which is required for genomic reprogramming and transcriptional activation in embryos. Recently, it has been shown that Nanog coordinates the organization of large transcription bodies during the maternal-to-zygotic transition. While live imaging of exogenous, fluorescently tagged Nanog has revealed important molecular behaviors, the physiological relevance of these experiments is unknown. Using a GEAR tagged Nanog, we investigated its endogenous behavior. When visualizing Nanog localization and dynamics by live imaging, we find fewer Nanog clusters that are notably smaller in comparison to exogenously introduced Nanog, further underscoring the physiological relevance of probing endogenous targets. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community.