Significance A mutation in myocilin is the most common known genetic cause of primary open-angle glaucoma (POAG). These mutations, which are dominant in nature, affect trabecular meshwork (TM) health and/or function and cause elevated intraocular pressure. Using in vitro human trabecular meshwork cells, an in vivo mouse model, and ex vivo human eyes, our study demonstrates the potential of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing in human myocilin-associated POAG. By disrupting the mutant myocilin gene and its function using CRISPR-Cas9, we were able to reduce associated endoplasmic reticulum stress, lower intraocular pressure, and prevent further glaucomatous damage in mouse eyes. We also show the feasibility of using the CRISPR-Cas9 system in cultured human eyes.