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Karina Luiza Dias Teixeira, John Doench, Gordon W Laurie; Genome-Wide CRISPR/Cas9 Screen Towards Identification of the Lacritin Homeostasis Receptor. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4705.
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Ocular tears refract 80% of light and are essential for epithelial homeostasis. 5 - 7% or more of the world’s population suffer from tear insufficiency associated with compromised visual acuity and chronic ocular surface inflammation. Lacritin, a mitogenic and antimicrobial glycoprotein found in tears yet deficient in dry eye, restores homeostasis in a mouse dry eye model, and in human corneal epithelial cells (HCET) subjected to dry eye-like IFN-γ and TNF-α stress. It does so by transiently stimulating autophagy to restore mitochondrial oxidative phosphorylation. Although we understand in general how lacritin works, there are many factors involved in lacritin pro-homeostatic signaling that remain unknown, including the signaling receptor - possibly a GPCR, and its interaction with coreceptor SDC1. Here we searched for and identified a candidate receptor using a forward genome-wide CRISPR/Cas9 screen.
HCET cells were transduced with the human Brunello CRISPR/Cas9 pooled library that comprises four sgRNAs targeting each of 19,114 human protein coding genes. Loss of homeostasis was modeled by introduction of fatal amounts of IFN-γ (1000U/mL) and TNF-α (100ng/mL) to search for cells incapable of rescue by lacritin 'N-94' synthetic peptide representing lacritin's C-terminal active domain. sgRNA's in cells that could no longer respond to N-94 were then identified by deep sequencing. Changes in abundance of perturbations from sample to sample were calculated using log-normalized data to generate log-fold-change (LFC) values displayed in volcano-plots with initial validation by shRNA knockdown.
The G protein coupled receptor 87 (GPR87) appears to be a strong candidate, showing its sgRNA enrichment in dead cells and depletion in alive cells, demonstrating the essentiality of this gene. Validation shRNA knockdown of GPR87 confirmed loss of responsiveness to N-94. Putative signaling mediators were also identified including key players in the Hippo pathway such as the transcription factor TEAD4. shRNA knockdown of TEAD4 expression also abrogated lacritin homeostasis activity.
This is the first genome-wide CRISPR/Cas9 sgRNA screen for gene products involved in lacritin-dependent restoration of homeostasis. Our main goal was identification of the signaling receptor for which GPR87 emerged as the top candidate.
This is a 2020 ARVO Annual Meeting abstract.
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