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.

Figure 1.

Overview of GFP₁₁ knock-in generation via CRISPR-Cas9-mediated HDR. Synthetic oligonucleotide primers are used to generate a DNA template. The DNA template enables the production of sgRNA through in vitro transcription. A Cas9/sgRNA ribonucleoprotein (RNP) complex is assembled in vitro. An ssDNA HDR repair template and the Cas9/sgRNA RNP complex are nucleofected into HEK 293 cells. GFP₁₁ knock-in cells can be analyzed by flow cytometry to assess the knock-in efficiency and enrich GFP-positive cells or be imaged by fluorescence microscopy to localize the tagged proteins.

Figure 2.

Example of an HDR repair template design. Top: An HDR template contains a 3-aa linker (black bold upper case), a GFP₁₁ coding sequence (green bold upper case), and two flanking homology arms for recombination (black lower case). The template is designed to integrate GFP₁₁ to the 3’ end of the HIST2H2BE gene immediately upstream of the stop codon (underlined lower case). Bottom: A region within the HIST2H2BE locus harboring a ~20 nucleotide sequence for the sgRNA site (purple underlined lower case).

Figure 3.

Example of a sgRNA design. (a) A DNA template eventually used for in vitro transcription of sgRNA (Step 5 in the 3.1.2 section). This template holds a T7 promoter, a gene-specific sequence, and a common sgRNA scaffold. (b) An overlapping PCR scheme for the synthesis of the DNA template. The DNA template is PCR-amplified using three common primers (T25, ML611, BS7) and a gene-specific primer. See Table 1 for the 5’ and 3’ primer sequences.

Figure 4.

A representative flow cytometry histogram of GFP₁₁ knock-in HEK 293 cells (green). To estimate the GFP₁₁ knock-in efficiency a the HIST2H2BE locus, GFP_1-10-expressing cells were used as a non-nucleofected control (gray). In this example, ~ 10% of the nucleofected cells are GFP-positive.

Figure 5.

A representative confocal image of GFP₁₁ knock-in HEK 293 cells. Endogenous H2B proteins are labeled with split GFP. The resulting reconstituted GFP signal displays the nuclear localization of Histone 2B. Scale bar, 20 μm.