Studying Endogenous Protein Dynamics with CRISPR-Mediated Tagging: Understanding Your Options
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Dr. Christopher Eggers received his Ph.D. in biochemistry and molecular biology from the University of California at San Francisco and then completed a postdoctoral fellowship at the Howard Hughes Medical Institute at UC San Diego. Since 2011, Dr. Eggers has been a Senior Research Scientist at Promega, where he has focused principally on the development of the NanoLuc® and NanoBiT® technologies to create new bioluminescent assays that simplify the measurement of protein dynamics.
Tuesday, June 4, 2019
An exciting application of CRISPR/Cas9-mediated gene editing is the ability to tag and measure endogenously expressed proteins while maintaining native expression levels and regulation. In this webinar, we will discuss available options for simple and efficient knock-in of a bioluminescent peptide tag to enable sensitive, homogeneous assays for measuring protein dynamics.
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CRISPR/Cas9 technology has revolutionized genome editing by offering a simple method to tag proteins at endogenous loci, facilitating the study of protein biology while maintaining proper transcriptional regulation, expression levels and stoichiometry with binding partners. By contrast, ectopic expression of tagged proteins can lead to a variety of overexpression artifacts, like mislocalization, aggregation or dysregulation of degradation. HiBiT, an 11-amino-acid bioluminescent peptide, represents an ideal tag for endogenous labeling due to its small size and large, linear dynamic range. In this webinar, we will highlight an efficient, cloning-free method for knock-in of HiBiT, as well as other protein tags, and we will discuss how these endogenously modified cells can be used in a variety of assay formats to study protein abundance, localization, modification and interactions. Additionally, we will discuss do-it-yourself approaches, the availability of pools and stable clones, and support for assay development.