A Practical Guide to CRISPR-Mediated Gene Tagging with a Bioluminescent Peptide

Using CRISPR-Cas9 technology, double-strand break (DSBs) can be created 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 will eliminate overexpression artifacts, mislocalization and aberrant signaling, providing a better understanding of native protein biology. However, many existing protein tags are large making CRISPR-mediated knock-in an inefficient process. In addition antibody-based detection methods and low sensitivity make many protein tags undesirable for use as endogenous tags. Recently, the HiBiT bioluminescent protein tagging system was developed, providing a tagging option that is amenable to CRISPR-mediated gene tagging due to the small 11 amino acid tag size, great sensitivity and simple antibody-free detection methods. In this webinar, we will walk you through a simple and efficient method for CRISPR-mediated HiBiT tagging that requires no molecular cloning steps, taking you from gene editing to assaying endogenous biology in as little as 24 hours.