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CRISPR-Cas9-Mediated Knock-In Approach to Insert the GFP11 Tag into the Genome of a Human Cell Line

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Fluorescent Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2564))

Abstract

The protocol in this chapter describes a method to label endogenous proteins using a self-complementing split green fluorescent protein (split GFP1-10/11) in a human cell line. By directly delivering Cas9/sgRNA ribonucleoprotein (RNP) complexes through nucleofection, this protocol allows for the efficient integration of GFP11 into a specific genomic locus via CRISPR-Cas9-mediated homology-directed repair (HDR). We use the GFP11 sequence in the form of a single-stranded DNA (ssDNA) as an HDR template. Because the ssDNA with less than 200 nucleotides used here is commercially synthesized, this approach remains cloning-free. The integration of GFP11 is performed in cells stably expressing GFP1-10, thereby inducing fluorescence reconstitution. Subsequently, such a reconstituted signal is analyzed using fluorescence flow cytometry for estimating knock-in efficiencies and enriching the GFP-positive cell population. Finally, the enriched cells can be visualized using fluorescence microscopy.

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Acknowledgments

We want to thank all members of the Kamiyama lab for critical comments on the manuscript; we particularly thank Melissa Inal and Kathy Bui for helpful discussion. This work was supported by an NIH R01 NS107558 (R.T. and D.K.). Ryo Tamura was supported by the Nakajima Foundation.

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Authors and Affiliations

  1. Department of Cellular Biology, University of Georgia, Athens, GA, USA

    Ryo Tamura & Daichi Kamiyama

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  1. Ryo Tamura
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  2. Daichi Kamiyama
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Correspondence to Daichi Kamiyama .

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Editors and Affiliations

  1. Institute of Molecular Plant Biology, ETH Zürich, Zürich, Switzerland

    Mayank Sharma

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Tamura, R., Kamiyama, D. (2023). CRISPR-Cas9-Mediated Knock-In Approach to Insert the GFP11 Tag into the Genome of a Human Cell Line. In: Sharma, M. (eds) Fluorescent Proteins. Methods in Molecular Biology, vol 2564. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2667-2_8

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  • DOI: https://doi.org/10.1007/978-1-0716-2667-2_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2666-5

  • Online ISBN: 978-1-0716-2667-2

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