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

Current protocols in stem cell biology (2020-07-07)
Rabea Dettmer, Ortwin Naujok
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ampicillin, anhydrous, 96.0-102.0% (anhydrous basis)
Sigma-Aldrich
Fetal Bovine Serum, USA origin, sterile-filtered, suitable for cell culture, suitable for hybridoma
Sigma-Aldrich
Trypsin-EDTA solution, 1 ×, sterile; sterile-filtered, BioReagent, suitable for cell culture, 0.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red
Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, 10×, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture