Design and Derivation of Multi‐Reporter Pluripotent Stem Cell Lines via CRISPR/Cas9n‐Mediated Homology‐Directed Repair

During the past decade, RNA-guided Cas9 nuclease from microbial clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) has become a powerful tool for gene editing of human pluripotent stem cells (PSCs). Using paired CRISPR/Cas9 nickases (CRISPR/Cas9n) it is furthermore possible to reduce off-target effects that may typically occur with traditional CRISPR/Cas9 systems while maintaining high on-target efficiencies. With this technology and a well-designed homology-directed repair vector (HDR), we are now able to integrate transgenes into specific gene loci of PSCs in an allele conserving way. In this protocol we describe CRISPR/Cas9n design and homology directed repair vector design, transfection of human pluripotent stem cells and selection and expansion of generated cell clones. © 2020 The Authors. Basic Protocol 1: Repair template design and CRISPR/Cas9n construction Basic Protocol 2: Transfection of human pluripotent stem cells by electroporation Basic Protocol 3: Genotyping of generated cell clones.