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CRISPR Manipulations in Stem Cell Lines

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Retinitis Pigmentosa

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2560))

Abstract

Insights into genome engineering in cells have allowed researchers to cultivate and modify cells as organoids that display structural and phenotypic features of human diseases or normal health status. The generation of targeted mutants is a crucial step toward studying the biomedical effect of genes of interest. Modified organoids derived from patients’ tissue cells are used as models to study diseases and test novel drugs. CRISPR-Cas9 technology has contributed to an explosion of advances that have the ability to edit genomes for the study of monogenic diseases and cancers. The generation of such mutants in human induced pluripotent stem cells (iPSCs) is of utmost importance as these cells carry the potential to be differentiated into any cell lineage. We describe recent developments that are broadening our understanding and extend DNA specificity, product selectivity, and fundamental capabilities. Furthermore, fundamental capabilities and remarkable advancements in basic research, biotechnology, and therapeutics development in cell engineering are detailed within this chapter. Using the CRISPR/Cas9 nuclease system for induction of targeted double-strand breaks, gene editing of target loci in iPSCs can be achieved with high efficiency. This chapter includes detailed protocols for the preparation of reagents to target loci of interest and transfection to genotype single cell-derived iPSC clones. Furthermore, we provide a protocol for the convenient generation of ribonucleoprotein (RNP) delivered directly to cells.

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Conflict of Interest

Stephen H. Tsang receives grant support from Abeona Therapeutics, Inc and Emendo. He is also the founder of Rejuvitas and is on the scientific and clinical advisory board for Nanoscope Therapeutics and Medical Excellence Capital.

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Authors and Affiliations

  1. Edward S. Harkness Eye Institute, Department of Ophthalmology, Columbia University Irving Medical Center, New York, NY, USA

    Ya-Ju Chang, Xuan Cui, Sarah R. Levi & Laura A. Jenny

  2. Jonas Children’s Vision Care and Bernard & Shirlee Brown Glaucoma Laboratory, New York, NY, USA

    Ya-Ju Chang, Xuan Cui, Sarah R. Levi & Laura A. Jenny

  3. Departments of Ophthalmology, Pathology & Cell Biology, Graduate Programs in Nutritional & Metabolic Biology and Neurobiology & Behavior, Columbia Stem Cell Initiative, New York, NY, USA

    Stephen H. Tsang

Authors
  1. Ya-Ju Chang
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  2. Xuan Cui
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  3. Sarah R. Levi
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  4. Laura A. Jenny
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  5. Stephen H. Tsang
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Corresponding author

Correspondence to Stephen H. Tsang .

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Editors and Affiliations

  1. Departments of Ophthalmology, Pathology & Cell Biology Graduate Programs in Nutritional & Metabolic Biology and Neurobiology & Behavior, Columbia Stem Cell Initiative, New York, NY, USA

    Stephen H. Tsang

  2. Department of Ophthalmology, Columbia University Medical Center, New York, NY, USA

    Peter M.J. Quinn

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Chang, YJ., Cui, X., Levi, S.R., Jenny, L.A., Tsang, S.H. (2023). CRISPR Manipulations in Stem Cell Lines. In: Tsang, S.H., Quinn, P.M. (eds) Retinitis Pigmentosa. Methods in Molecular Biology, vol 2560. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2651-1_23

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  • DOI: https://doi.org/10.1007/978-1-0716-2651-1_23

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2650-4

  • Online ISBN: 978-1-0716-2651-1

  • eBook Packages: Springer Protocols

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