Methods in molecular biology
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Gene Editing in Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible CRISPR-Cas9 System.
Induced pluripotent stem cells (iPSCs) generated from patients are a valuable tool for disease modelling, drug screening, and studying the functions of cell/tissue-specific genes. However, for this research, isogenic iPSC lines are important for comparison of phenotypes in the wild type and mutant differentiated cells generated from the iPSCs. The advent of gene editing technologies to correct or generate mutations helps in the generation of isogenic iPSC lines with the same genetic background. ⋯ An iPSC line with drug inducible Cas9 expression from the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus offers a controllable expression of Cas9 with robust gene editing. Here, we describe a stepwise protocol for the generation and characterization of such an iPSC line (AAVS1-PDi-Cas9 iPSC) with a doxycycline (dox)-inducible Cas9 expression cassette from the AAVS1 safe harbor site and efficient editing of target genes with lentiviral vectors expressing gRNAs. This approach with a tunable Cas9 expression that allows investigating gene functions in iPSCs or in the differentiated cells can serve as a versatile tool in disease modelling studies.