Genome-Scale CRISPR Screens Identify Human Pluripotency-Specific Genes
Robert J. Ihry,
Max R. Salick,
Daniel J. Ho,
Marie Sondey,
Sravya Kommineni,
Steven Paula,
Joe Raymond,
Beata Henry,
Elizabeth Frias,
Qiong Wang,
Kathleen A. Worringer,
Chaoyang Ye,
Carsten Russ,
John S. Reece-Hoyes,
Robert C. Altshuler,
Ranjit Randhawa,
Zinger Yang,
Gregory McAllister,
Gregory R. Hoffman,
Ricardo Dolmetsch,
Ajamete Kaykas
Affiliations
Robert J. Ihry
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; Corresponding author
Max R. Salick
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; Insitro, South San Francisco, CA 94080, USA
Daniel J. Ho
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Marie Sondey
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; Abbvie, Cambridge, MA 02139, USA
Sravya Kommineni
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; Casma Therapeutics, Cambridge, MA 02139, USA
Steven Paula
Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Joe Raymond
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Beata Henry
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Elizabeth Frias
Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Qiong Wang
Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Kathleen A. Worringer
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Chaoyang Ye
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; Blueprint Medicines, Cambridge, MA 02139, USA
Carsten Russ
Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
John S. Reece-Hoyes
Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Robert C. Altshuler
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Ranjit Randhawa
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; Axcella Health, Cambridge, MA 02139, USA
Zinger Yang
Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; University of Massachusetts Medical School, Worcester, MA 01655, USA
Gregory McAllister
Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; Sana Biotechnology, Cambridge, MA 02139, USA
Gregory R. Hoffman
Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; Sana Biotechnology, Cambridge, MA 02139, USA
Ricardo Dolmetsch
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
Ajamete Kaykas
Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA; Insitro, South San Francisco, CA 94080, USA; Corresponding author
Summary: 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. : Ihry et al. develop a CRISPR/Cas9 genetic screening platform for hPSCs that enables unbiased genome-scale genetic screening. The platform exhibits high performance and accurately detects the dropout of essential genes. Furthermore, proof-of-concept screens exploit hPSC-specific phenotypes to identify regulators of fitness, survival after single-cell dissociation, and pluripotency. Keywords: CRISPR genome-wide screening, human pluripotent stem cells, iPSC, hESC, PAWR, PMAIP1, DDR
