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

Abstract

The protocol in this chapter describes a method to label endogenous proteins using a self-complementing split green fluorescent protein (split GFP_1-10/11) 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.

Keywords: CRISPR; Cas9; GFP1-10; GFP11; Homology-directed repair; Split GFP.