Abstract
Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.
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Acknowledgments
This work was supported by research grants from the American Heart Association 17MERIT33610009, National Institutes of Health (NIH) R01 HL113006, R01 HL123968, R01 HL141851, UH3 TR002588 (JCW), R01 HL 126527 (LSQ), U01 EB021240 (LSQ), and JSPS Overseas Research Fellowship (MN). The CRISPRi iPSC line (CRISPRi Gen2C) was kindly provided by Conklin lab (Gladstone Institute) [8].
Disclosures
JCW is a cofounder of Khloris Biosciences but has no competing interests, as the work presented here is completely independent. LSQ is a cofounder of Refuge Biotechnologies but has no competing interests, as the work presented here is completely independent.
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Nishiga, M., Qi, L.S., Wu, J.C. (2021). CRISPRi/a Screening with Human iPSCs . In: Yoshida, Y. (eds) Pluripotent Stem-Cell Derived Cardiomyocytes. Methods in Molecular Biology, vol 2320. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1484-6_23
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DOI: https://doi.org/10.1007/978-1-0716-1484-6_23
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