Figure 5.

Overview of the LacI-EGFP reporter and feasibility studies in human cells. (A) Schematic of the LacI-EGFP reporter carrying EGFP-FKBP regulated by Lac repressor. EF-1α, Human elongation factor-1α (EF-1α) promoter; multiple target sites, target sites for sgRNA; LacI, DNA-binding transcriptional repressor LacI; pA, polyadenylation signal sequence; oo, double lac operator; CMV, cytomegalovirus promoter; FKBP, FKBP12-derived destabilizing domain. (B) Enhancement of signal-to-noise ratio of EGFP with EGFP-FKBP. MFI (mean fluorescence intensity) of EGFP was measured and fold change was calculated as the MFI of the non-functional Lac repressor over that of the wild type repressor (n = 3; mean ± SD; ***P < 0.001; unpaired t-test). EGFP-FKBP reduced EGFP background and increase EGFP signal compared to EGFP in the LacI-EGFP reporter. (C) Feasibility of using the LacI-EGFP reporter to measure the cleavage efficiency of sgRNAs. The px459 (sgRNA plus Cas9) and LacI-EGFP reporter plasmids were transfected into HEK293T cells. The MFI of EGFP was measured and the fold change was calculated as the MFI of the experimental groups divided by that of the control. (D) Correlation of the cleavage efficiency of sgRNAs measured using the LacI-luciferase reporter and LacI-EGFP reporter assays. Pearson correlation coefficient r = 0.9218, P = 0.0070, (n = 3; mean ± SD).