A Practical Guide to CRISPR-Mediated Gene Tagging with a Bioluminescent Peptide
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Dr. Schwinn is a member of the cell biology team within the Advanced Technologies Group at Promega where she has helped develop luciferase-based technologies for studying intracellular protein interactions, abundance, and post-translational modifications. Most recently, she has focused on combining these technologies with CRISPR/Cas9 to study protein dynamics at the endogenous level. Prior to joining Promega, Dr. Schwinn earned her doctorate in Biochemistry from the University of Wisconsin-Madison in the lab of Dr. Hector F. DeLuca, and she received post-doctoral training from Dr. Donna M. Peters in the Department of Pathology and Laboratory Medicine.
Tuesday, October 24, 2017
CRISPR/Cas9 gene editing has emerged as a powerful tool for making precise genomic modifications. Although often used to create gene knockouts, the technology also enables targeted knock-in of a specific sequence. An exciting application is the endogenous tagging of proteins expressed under native regulatory conditions. In this webinar, we will detail a simple and efficient cloning-free method for CRISPR-mediated endogenous gene tagging, focusing on knock-in of the sensitive, bioluminescent HiBiT tag.
CRISPR-Cas9 technology creates double-strand breaks (DSBs) at almost any genomic location of interest. Following the DSB, cellular repair machinery can either undergo non-homologous end joining (NHEJ) creating knockouts, or, if a donor DNA template is provided, the cell can undergo homology directed repair (HDR) to knock-in a precise modification or insertion. Often researchers may want to knock-in a tag sequence, enabling further study of protein biology under endogenous regulatory conditions. Compared to ectopic expression of a tagged protein, endogenous locus expression eliminates artifacts of overexpression, mislocalization and aberrant signaling to provide a better understanding of native protein biology. Many existing protein tags are large, which makes CRISPR-mediated knock-in inefficient. Antibody-based detection methods and low sensitivity make many protein tags undesirable for use as endogenous tags. We recently developed the HiBiT bioluminescent protein tagging system, which provides a tagging option that is amenable to CRISPR-mediated gene tagging. The HiBiT tagging system contains a tag of only 11 amino acids with great sensitivity rendering simple antibody-free protein detection. In this webinar, we will demonstrate an efficient method for CRISPR-mediated HiBiT tagging that requires no molecular cloning steps so that you can progress from gene editing to assaying endogenous biology in as little as 24 hours.