The green fluorescent protein (GFP)-based reporter system has been widely harnessed as a quick quantitative activity assessment method for characterizing CRISPR-Cas via flow cytometry. However, due to the small size (738 nt) of the GFP coding sequence, the targeting sites for certain CRISPR-Cas are greatly restricted. To address this, here we developed a GFP tagged polycistronic reporter system to determine the activity of CRISPR-Cas in human cells. Specifically, the system contains the herpes simplex virus thymidine kinase (TK) gene, bacterial neomycin phosphotransferase (Neo) gene, and green fluorescent protein (GFP), named TNG gene, with a coding sequence of 2,577 nt. To investigate its performance, we generated a human cell line harboring the TNG expression cassette at the AAVS1 locus, and then we tested it with different Cas orthologs (SaCas9, St1Cas9, and AsCas12a). Our results demonstrated that using the TNG reporter system greatly expands the targeting site selection (3- to 13-fold) with CRISPR-Cas genome editing. The study therefore reports an additional method for the characterization of CRISPR-Cas technology.
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