Abstract
Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine and disease modeling. Here, we present a protocol for establishing edited hPSC cell lines utilizing visualized orthogonal selective reporters. We describe steps for constructing plasmids, carrying out cell culture and electroporation, as well as performing drug-fluorescent dual enrichment, clone screening, and cell line characterization. This protocol facilitates the achievement of single-base homozygous mutations and reporter knockins, offering a reliable approach for precision genome editing.