Protocol for scarless genome editing of human pluripotent stem cell based on orthogonal selective reporters

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.