Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1961; p. 185
• Main Authors: Snijders, Kirsten E, Cooper, James D, Vallier, Ludovic, Bertero, Alessandro
• Format: Journal Article
• Language: English
• Published: United States 2019
• Subjects: CRISPR-Cas Systems - genetics; Gene Knockout Techniques - methods; Humans; Pluripotent Stem Cells - metabolism; RNA, Guide, CRISPR-Cas Systems - genetics; Inducible knockout; CRISPR/Cas9; Human pluripotent stem cells; sgRNA; AAVS1; Tetracycline
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9170-9_12

The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based OPTimized inducible gene KnockOut method (OPTiKO) for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single guide RNAs (sgRNAs) that drive Cas9 activity. In order to ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease.