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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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
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Publisher Name: Humana, New York, NY
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