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Medientyp: E-Artikel Titel: Design and Derivation of Multi‐Reporter Pluripotent Stem Cell Lines via CRISPR/Cas9n‐Mediated Homology‐Directed Repair Beteiligte: Dettmer, Rabea; Naujok, Ortwin Erschienen: Wiley, 2020 Erschienen in: Current Protocols in Stem Cell Biology Sprache: Englisch DOI: 10.1002/cpsc.116 ISSN: 1941-7322; 1938-8969 Schlagwörter: Cell Biology ; Developmental Biology ; General Medicine Entstehung: Anmerkungen: Beschreibung: <jats:title>Abstract</jats:title><jats:p>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.</jats:p><jats:p><jats:bold>Basic Protocol 1</jats:bold>: Repair template design and CRISPR/Cas9n construction</jats:p><jats:p><jats:bold>Basic Protocol 2</jats:bold>: Transfection of human pluripotent stem cells by electroporation</jats:p><jats:p><jats:bold>Basic Protocol 3</jats:bold>: Genotyping of generated cell clones</jats:p>