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Human in vitro model systems of diabetes are critical to both study disease pathophysiology and offer a platform for drug testing. We have generated a set of tools in the human β-cell line EndoC-βH1 that allows the efficient and inexpensive characterization of β-cell physiology and phenotypes driven by disruption of candidate genes. First, we generated a dual reporter line that expresses a preproinsulin-luciferase fusion protein along with GCaMP6s. This reporter line allows the quantification of insulin secretion by measuring luciferase activity and calcium flux, a critical signaling step required for insulin secretion, via fluorescence microscopy. Using these tools, we demonstrate that the generation of the reporter human β-cell line was highly efficient and validated that luciferase activity could accurately reflect insulin secretion. Second, we used a lentiviral vector carrying the CRISPR-Cas9 system to generate candidate gene disruptions in the reporter line. We also show that we can achieve gene disruption in ~90% of cells using a CRISPR-Cas9 lentiviral system. As a proof of principle, we disrupt the β-cell master regulator, PDX1, and show that mutant EndoC-βH1 cells display impaired calcium responses and fail to secrete insulin when stimulated with high glucose. Furthermore, we show that PDX1 mutant EndoC-βH1 cells exhibit decreased expression of the β-cell-specific genes MAFA and NKX6.1 and increased GCG expression. The system presented here provides a platform to quickly and easily test β-cell functionality in wildtype and cells lacking a gene of interest., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)