Intracellular
signaling pathways are mediated by changes in protein
abundance and post-translational modifications. A common approach
for investigating signaling mechanisms and the effects induced by
synthetic compounds is through overexpression of recombinant reporter
genes. Genome editing with CRISPR/Cas9 offers a means to better preserve
native biology by appending reporters directly onto the endogenous
genes. An optimal reporter for this purpose would be small to negligibly
influence intracellular processes, be readily linked to the endogenous
genes with minimal experimental effort, and be sensitive enough to
detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino
acids) capable of producing bright and quantitative luminescence through
high affinity complementation (<i>K</i><sub>D</sub> = 700
pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9,
we demonstrate that HiBiT can be rapidly and efficiently integrated
into the genome to serve as a reporter tag for endogenous proteins.
Without requiring clonal isolation of the edited cells, we were able
to quantify 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 HiBiT to directly correlate HIF1α levels with
the hydroxyproline modification that mediates its degradation. These
results demonstrate the ability to efficiently tag endogenous proteins
with a small luminescent peptide, allowing sensitive quantitation
of the response dynamics in their regulated expression and covalent
modifications
