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Fig. 2 | BMC Genomics

Fig. 2

From: Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing

Fig. 2

The frame-shift CFP (FsCFP) reporter successfully distinguished genome editing events of different targeting efficiencies, with very low noise signals. See also Additional file 1: Figure S2 and S3 for the establishment of the detection gatings, and S4 for the intensity of background CFP signal. a Genome editing was performed on a target sequence derived from the RRS gene of Chinese hamster. The corresponding gRNA is not expected to react with the essential RRS gene in the HEK293T cells, which is the host cell for the editing. The sequence of the wildtype gRNA and the mutant gRNA are presented on top, where the two mutated nucleotides in the center of the mutRRS-gRNA are indicated by red color. On the bottom, two representative flow charts show the HEK293T cells transduced with the FsCFP with the RRS-targeting sequence and the wildtype or mutatnt RRSgRNA-Cas9. Among the cells expressing both the mCherryFP and GFP, which are the markers for the FsCFP and the gRNA-Cas9, repectively, a subset of them also exhibited detectable CFP signals. b Similar to A, except that a target sequence derived from human VEGF and the correponding gRNA are used. c Representative flow charts of cells with VEGF-FsCFP and either a non-targeting sequence gRNA (ns-gRNA) and Cas9, or just GFP alone without gRNA and Cas9. In both the cases, the ratio of CFP-positive cells was close to or lower than 0.01%. d Quantification of CFP-positive events detected in mCherryFP and GFP double positive cells with different gRNAs as shown in a, b and c. The mean value was calculated from independent repeats (n = 4 for RRS and mutRRS; n = 3 for the others). The error of means were shown after the +/− sign

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