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CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent Stem Cells

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Induced Pluripotent Stem Cells and Human Disease

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2549))

Abstract

Genetically encoded fluorescent biosensors (GEFBs) enable researchers to visualize and quantify cellular processes in live cells. Induced pluripotent stem cells (iPSCs) can be genetically engineered to express GEFBs via integration into the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus. This can be achieved using CRISPR/Cas ribonucleoprotein targeting to cause a double-strand break at the AAVS1 locus, which subsequently undergoes homology-directed repair (HDR) in the presence of a donor plasmid containing the GEFB sequence. We describe an optimized protocol for CRISPR/Cas-mediated knock-in of GEFBs into the AAVS1 locus of human iPSCs that allows puromycin selection and which exhibits negligible off-target editing. The resulting iPSC lines can be differentiated into cells of different lineages while retaining expression of the GEFB, enabling live-cell interrogation of cell pathway activities across a diversity of disease models.

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Acknowledgments

This work was supported by the Merridew Foundation and the Batten Disease Support and Research Association (Australia). pMitoTimer was a gift from Zhen Yan (Addgene plasmid # 52659; http://n2t.net/addgene:52659; RRID:Addgene_52659). pmTurquoise2-Tubulin was a gift from Dorus Gadella (Addgene plasmid # 36202; http://n2t.net/addgene:36202; RRID:Addgene_36202). pMRX-IP-GFP-LC3-RFP-LC3ΔG was a gift from Noboru Mizushima (Addgene plasmid # 84572; http://n2t.net/addgene:84572; RRID:Addgene_84572). Figures created with BioRender.com.

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Authors and Affiliations

  1. Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, TAS, Australia

    David Stellon, Jana Talbot, Sueanne Chear, James C. Vickers, Anna E. King & Anthony L. Cook

  2. Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia

    Minh Thuan Nguyen Tran, Mohd Khairul Nizam Mohd Khalid & Alex W. Hewitt

  3. Department of Anatomy and Physiology, University of Melbourne, Parkville, VIC, Australia

    Alice Pébay

  4. Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Parkville, VIC, Australia

    Alice Pébay

Authors
  1. David Stellon
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  2. Minh Thuan Nguyen Tran
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  3. Jana Talbot
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  4. Sueanne Chear
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  5. Mohd Khairul Nizam Mohd Khalid
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  6. Alice Pébay
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  7. James C. Vickers
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  8. Anna E. King
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  9. Alex W. Hewitt
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  10. Anthony L. Cook
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Corresponding authors

Correspondence to David Stellon or Anthony L. Cook .

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Editors and Affiliations

  1. Ottawa, ON, Canada

    Kursad Turksen

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Stellon, D. et al. (2021). CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent Stem Cells. In: Turksen, K. (eds) Induced Pluripotent Stem Cells and Human Disease. Methods in Molecular Biology, vol 2549. Humana, New York, NY. https://doi.org/10.1007/7651_2021_422

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  • DOI: https://doi.org/10.1007/7651_2021_422

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2584-2

  • Online ISBN: 978-1-0716-2585-9

  • eBook Packages: Springer Protocols

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