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Updated: Sep 16, 2025
Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation
Published on: February 28, 2025
Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene
Yao Zhang1, Hao Yang2, Yuan Yang3
1Institute of Neurological and Psychiatric Disorders, Shenzhen Bay Laboratory, Shenzhen 518132, China; School of Medicine, Shenzhen Campus of Sun Yat-sen University, Sun Yat-sen University, Shenzhen 518107, China.
Abstract:
CRISPR-Cas9 is widely used for genome editing. However, Cas9 silencing occurs during the directed differentiation of induced pluripotent stem cells (iPSCs), even when it is inserted into the safe harbor locus. Here, we generate iPSC-Cas9-EGFP using selection by essential gene exon knockin technology. We describe steps for inserting the Cas9-EGFP into exon 9 of GAPDH, bypassing Cas9 silencing. We then detail procedures for Cas9 function validation. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.
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