Abstract
Human pluripotent stem cells (hPSCs) generate a variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited by technical challenges. We developed a scalable and renewable Cas9 and sgRNA-hPSC library in which loss-of-function mutations can be induced at will. Our inducible mutant hPSC library can be used for multiple genome-wide CRISPR screens in a variety of hPSC-induced cell types. As proof of concept, we performed three screens for regulators of properties fundamental to hPSCs: their ability to self-renew and/or survive (fitness), their inability to survive as single-cell clones, and their capacity to differentiate. We identified the majority of known genes and pathways involved in these processes, as well as a plethora of genes with unidentified roles. This resource will increase the understanding of human development and genetics. This approach will be a powerful tool to identify disease-modifying genes and pathways.
Highlights
Human pluripotent stem cells can be used to generate a wide variety of disease-relevant cell types and have the potential to improve the translation of preclinical research by enhancing disease models
We addressed these technical issues by systematically tailoring the CRISPR/Cas9 system for Human pluripotent stem cells (hPSCs) (Ihry et al, 2018)
We identified genes required for singlecell cloning. hPSCs have a poor survival rate after dissociation to single cells, which is detrimental for genome engineering
Summary
Human pluripotent stem cells (hPSCs) can be used to generate a wide variety of disease-relevant cell types and have the potential to improve the translation of preclinical research by enhancing disease models. The CRISPR/Cas system is the genetic screening tool of choice because it can efficiently cause loss-of-function alleles (Jinek et al, 2012; Cong et al, 2013; Mali et al, 2013). In genetically intact hPSCs, the only genome-scale CRISPR screen to date used methods developed for cancer cells, suffered from technical issues, had poor performance, and identified few developmentally relevant genes (Hart et al, 2014; Shalem et al, 2014). We addressed these technical issues by systematically tailoring the CRISPR/Cas system for hPSCs (Ihry et al, 2018). This allowed us to conduct multiple independent screens with the same cell library
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