Abstract
Purpose :
To validate a simple, sensitive, and multifunctional endogenous protein tagging approach that enables small molecule/genetic high-throughput screens (HTS) and the probing of ocular disease-related biology.
Methods :
CRISPR/Sp.Cas9 engineering was used to introduce an 11 amino acid luminescent peptide tag (HiBiT) onto proteins associated with age-related macular degeneration (AMD) - complement factor H (CFH) and high temperature requirement A1 (HtrA1) in immortalized and induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial (RPE) cells. We appended this same tag also onto pigment epithelium-derived factor (PEDF), a cytoprotective protein produced in the retina. Editing was verified by genomic DNA analysis, short interfering RNA, and HiBiT blotting. Native quaternary structure was determined by size-exclusion chromatography (SEC). Small molecule HTS was performed on HtrA1 HiBiT cells, yielding potential enhancing compounds. These compounds were further examined for mechanism of action and via HiBiT assays, qPCR, and label-free proteomics.
Results :
CRISPR editing resulted in editing rates of 55-88%. siRNA verified the specificity of the tagging. Denaturing HiBiT blotting demonstrated a single tagged species for HtrA1 and PEDF (~50 kDa), and two species for CFH (full length and FHL-1). However, SEC followed by a HiBiT assay demonstrated the formation of higher order species (~400 kDa) under native conditions in CFH and HtrA1 media samples. Small molecule HTS on HtrA1 HiBiT cells identified compounds that significantly enhanced secreted and intracellular HtrA1 levels, which was confirmed upon follow up and secondary assays.
Conclusions :
HiBiT tagging serves as a simple and effective method to monitor endogenous protein production, native conformation/size and regulation. HiBiT cell lines can be used to easily and quickly perform HTS, identifying new lead small molecule regulators of retinal degeneration-associated proteins. We envision that this strategy can be used to gain insight into many disease-associated ocular proteins and identify new small molecule or genetic therapeutics for vision disorders.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.