CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Recorded On: 02/07/2018
The effects of synthetic compounds on signaling pathways are often evaluated using overexpressed genetic reporters. It is now possible with CRISPR/Cas9 to better preserve native biology by appending reporters directly onto the endogenous genes. For this purpose, we introduced the HiBiT peptide (11 amino acids) as a small reporter tag capable of producing bright and quantitative luminescence through complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). The small size of HiBiT minimally alters protein structure, while the luminescent assay provides sensitive analysis at very low expression levels. Using CRISPR/Cas9, we demonstrated that HiBiT can be rapidly and efficiently integrated into the genome to serve as a quantitative tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to determine changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used energy transfer from HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These assay methods allowed dynamics in protein abundance and covalent modifications to be assessed within 24-48 hours of introducing synthetic oligonucleotides together with Cas9 into the cells, thus circumventing the prerequisite for molecular cloning.
Keith Wood is Head of Research, Advanced Technologies and Senior Research Fellow at Promega Corporation. Widely regarded for his work in bioluminescence, he currently leads a cross‐disciplinary team focused on long‐range innovation in biochemical and cellular research. His current research centers on the development of bioanalytical capabilities, including novel bioluminescent chemistries, intracellular detection technologies, and efficient isolation methods for protein and drug complexes. He has authored over 64 journal articles and book chapters and is an inventor for 164 issued and 124 pending U.S. and foreign patents. Keith received his Ph.D. in Biochemistry at University of California‐San Diego, where he also performed post‐doctoral research before joining Promega in 1990.