June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Genome-Wide CRISPR/Cas9 Screen Towards Identification of the Lacritin Homeostasis Receptor
Author Affiliations & Notes
  • Karina Luiza Dias Teixeira
    Cell Biology, University of Virginia, Charlottesville, Virginia, United States
  • John Doench
    MIT and Harvard, Broad Institute, Massachusetts, United States
  • Gordon W Laurie
    Cell Biology, University of Virginia, Charlottesville, Virginia, United States
    Ophthalmology, University of Virginia, Virginia, United States
  • Footnotes
    Commercial Relationships   Karina Luiza Dias Teixeira, None; John Doench, None; Gordon Laurie, F; TearSolutions Inc. P; UVa Licensing and Ventures Group; (F)
  • Footnotes
    Support  EY024327, EY026171, UVA Pinn Scholar Award, and an unrestricted gift from TearSolutions, Inc,
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4705. doi:
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    • Get Citation

      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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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