Combining CRISPR/Cas9-mediated knockout with genetic complementation for in-depth mechanistic studies in human ES cells. (15th January 2019)

• Record Type: Journal Article
• Authors: Wang, Zheng; Zhang, Yan; Lee, Yu-Wei; Ivanova, Natalia B
• Is Part Of: Biotechniques. Volume 66:Number 1(2019)
• Page Start: 23
• Page End: 27
• Publication Date: 2019-01-15
• Subjects: CRISPR/Cas9 mutagenesis -- human embryonic stem cells -- pluripotency -- self-renewal; Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/btn-2018-0115
• Languages: English
• ISSNs: 0736-6205

Abstract

Gene regulatory networks that control pluripotency of human embryonic stem cells (hESCs) are of considerable interest for regenerative medicine. RNAi and CRISPR/Cas9 technologies have allowed the identification of hESC regulators on a genome-wide scale. However, these technologies are ill-suited for mechanistic studies because knockdown/knockout clones of essential genes do not grow in culture. We have developed a genetic rescue strategy that combines CRISPR/Cas9-mediated knockout with TALEN-mediated integration of a doxycycline-inducible rescue transgene into a constitutive AASV1 locus. The resulting rescue clones are stable in culture, allow modulation of the rescue transgene dosage by titration of doxycycline in the media and can be combined with various molecular assays, thus providing mechanistic insights into gene function in a variety of cellular contexts.