Genome-scale CRISPR-Cas9 knockout coupled with single-cell RNA sequencing (scRNA-seq) has been used to identify function-related genes. However, this method may knock out too many genes, leading to low efficiency in finding genes of interest. Insulin secretion is controlled by several electrophysiological events, including fluxes of KATP depolarization and K+ repolarization. It is well known that glucose stimulates insulin secretion from pancreatic β-cells, mainly via the KATP depolarization channel, but whether other nutrients directly regulate the repolarization K+ channel to promote insulin secretion is unknown. Here, we used a system involving CRISPR-Cas9-mediated knockout of all 83 K+ channels and scRNA-seq in a pancreatic β cell line to identify genes associated with insulin secretion. The expression levels of insulin genes were significantly increased after all-K+ channel knockout. Furthermore, Kcnb1 and Kcnh6 were the two most important repolarization K+ channels for the increase in high-glucose-dependent insulin secretion that occurred upon application of specific inhibitors of the channels. Kcnh6 currents, but not Kcnb1 currents, were reduced by one of the amino acids, lysine, in both transfected cells, primary cells and mice with β-cell-specific deletion of Kcnh6. In conclusion, our function-related CRISPR screen with scRNA-seq identifies Kcnh6 as a lysine-specific channel.Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.