Generation of isogenic iPSC lines through CRISPR/Cas9-Mediated Targeting

This protocol describes the generation of genetically modified human pluripotent stem cell (hPSC) lines using CRISPR/Cas9 genome editing. Guide RNAs are cloned into the pX330 Cas9 plasmid and co-delivered with targeting vectors into hPSCs. Following transfection, cells are cultured under supportive conditions to allow recovery and clonal outgrowth. Individual colonies are manually picked, expanded, and screened for successful targeting.