Key result
The TNG reporter system greatly expanded targeting site selection by 3- to 13-fold for CRISPR-Cas genome editing.
Why the study?
Due to the small size of the GFP coding sequence, targeting sites for certain CRISPR-Cas systems are greatly restricted in standard GFP-based reporter systems.
Effect estimate: 3- to 13-fold
The TNG reporter system expands targeting site selection 3- to 13-fold for characterizing CRISPR-Cas activity in human cells.
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 ( T K ) gene, bacterial neomycin phosphotransferase ( N eo ) gene, and green fluorescent protein ( G FP ), 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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Yang et al. (2022) studied this question. GFP tagged polycistronic reporter system (TNG) was evaluated on Targeting site selection expansion (3- to 13-fold). The TNG reporter system greatly expanded targeting site selection by 3- to 13-fold for CRISPR-Cas genome editing.
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