ABSTRACT INTRODUCTION Primary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterised by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. This study sought to investigate the morphological and transcriptional consequences of perturbation of key genes at IOP loci in trabecular meshwork cell (TMC); the cellular regulators of IOP. We aimed to identify genetic and morphological variation that can be attributed to TMC dysfunction and raised IOP in POAG. METHODS 62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescent staining of key organelles, was coupled with high-throughput microscopy for single-cell morphological analysis using CellProfiler image analysis. RESULTS Across many of the individual gene knockouts scRNA-seq highlighted genes relating to matrix metalloproteinases and interferon-induced proteins. Our work has prioritised genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1 . Three genetic networks of gene knockouts with similar transcriptomic profiles were identified ( ABO / CAV1 / MYOC , ANGPT2 / PKHD1 / TNS1 / TXNRD2 , and CAPZA1 / KALRN / LMO7 / PLEKHA7 / GNB1L / TEX41 ), suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, whilst knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. CONCLUSION High throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma