Optimization and Initiation of a Genome‐Wide Forward Genetic Death Screen for the Lacritin Receptor Complex using the Brunello CRISPR/Cas 9 sgRNA Library

Ocular tears refract 80% of light and are essential for epithelial homeostasis. 7% of the world's population suffer from tear insufficiency associated with compromised visual acuity and chronic ocular surface inflammation. Lacritin, a glycoprotein constituent of tears, restores homeostasis in a mouse dry eye model, and in human corneal epithelial cells (HCET) subjected to dry eye‐like IFN‐g and TNF‐a stress. It does so by transiently stimulating autophagy and restoring oxidative phosphorylation. Lacritin also promotes basal tearing by increasing the sensitivity of ion channel TRPM8 on corneal sensory nerves to tear drying. Less well understood is the receptor complex, likely inclusive of a GPCR ‐ based on pertussis toxin and MK912 sensitivity, active N‐24, but not inactive C‐25, lacritin truncation mutant inhibition (64%) of 125 I‐iodoclonidine binding to purified alpha‐2C adrenergic receptor (ADRA2C), and function loss after pooled siRNA knockdown of ADRA2C. ADRA2C is known to complex with ADRA2A, which in turn can modulate TRPM8. Yet, lacritin is unlike any other ADRA2C agonist, requires prior binding to heparanase modified syndecan‐1 (SDC1), failed to inhibit 125 Iiodoclonidine binding to ADRA2C and did not activate a beta‐annexin based ADRA2C Tango assay. Goal Our goal is to identify the lacritin GPCR(s) using a forward genome‐wide CRISPR/Cas9 screen in HCET cells stressed with interferon‐gamma (IFNG) and tumor necrosis factor (TNF). Methods Initial experiments took advantage of GFP expressing pRosetta to establish optimal HCET viral infection and polybrene concentration. Next, we optimized HCET infection with the Brunello human CRISPR/Cas9 lentiviral library (Addgene #73179) and selection with puromycin, followed by INFG/TNF. For the latter, Alamar Blue and Colony Assays such to identify INFG/TNF levels that killed all cells yet made possible recovery by lacritin using the N‐94 synthetic peptide representing lacritin's C‐terminal active domain. Deep sgRNA sequencing will be performed on genomic DNA from cells stressed with INFG/TNF without or with N‐94. Results For HCET cells, the optimal polybrene concentration was 4 mg/ml that yielded an infection efficiency of ~97% as determined by FACS. The optimal volume of Brunello lentiviral library to obtain 30–50% of infection efficiency was 200 μl. 2 μg/ml was best for puromycin, 1000 U/ml for INFG, 100 ng/ml for TNF and 1000 nM for N‐94 recovery of ~50% of cells. The library consists of 76,441 sgRNAs targeting 19,114 human genes (including ADRA2C, ADRA2A, TRPM8 and SDC1) plus 1000 non‐targeting sgRNA controls. The forward screen is now underway. Support or Funding Information NIH R01 EY024327 NIH R01 EY026171 Uva Pinn Scholar Award This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .