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. 2022 Oct 18;13:6167. doi: 10.1038/s41467-022-33287-9

Fig. 3. Genetic control elements added to maximize the expression level of the target gene.

Fig. 3

Schematic illustrations depicting individual experiments exhibit only plasmid constructs that were different from P1–P4 in Fig. 1c. Additional genetic control elements were incorporated into the crisprTF promoters (a–d). Experimental groups (+) were transiently transfected with four or five plasmids (P1–P4 or P1–P5), whereas negative controls (−) were transfected without P1. a Synergistic activation mediator (SAM). dCas9-VPR was combined with SAM, another strong synthetic transcriptional activator. Two-tailed paired Student’s t test was performed to compare two P1(+) groups with the same number of gRNA BS (with the 8× BS operator, MS2H gRNA vs. control gRNA, p = 0.0003; with the 16× BS operator, MS2H gRNA vs. control gRNA, p = 0.0023) b Dual gRNA transcriptional units (TUs). An extra gRNA10 TU was added to increase gRNA expression (two-tailed paired Student’s t test; with the 8× BS operator, dual gRNA vs. single gRNA, p = 0.0278; with the 16× BS operator, dual gRNA vs. single gRNA, p = 0.0090). c An additional 2× nuclear localizing sequence (NLS) in the crisprTF was added at the 5′-end of dCas9-VPR (two-tailed paired Student’s t test; with the 8× BS operator, 2× NLS vs. control, p = 0.0055; with the 16× BS operator, 2× NLS vs. control, p = 0.0034). d A synthetic intron (SI) was added at the 5′ UTR of the target gene. The SI was incorporated into four synthetic operators of the gRNA4, 7, and 10 series (4×, 8×, 12×, and 16× BS), respectively. Red solid bars represent experimental groups (+); paired red hollow bars represent corresponding control groups without gRNA (−) (one-way ANOVA with multiple comparisons corrected by Dunnett test for each gRNA series with the same EF1α and CMV control groups; CMV, gRNA4 P1(+) 8×, 12×, and 16× BS with SI, gRNA7 P1(+) 8×, 12×, and 16× BS with SI, and gRNA10 P1(+) 8×, 12×, and 16× BS with SI vs. EF1α: p < 0.0001, p = 0.0003, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0006, p < 0.0001, and p < 0.0001, respectively). All data represent the mean ± SD (n ≥ 3). For increased expression (marked in black): *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; for decreased expression (marked in blue): **p < 0.01; ND not detected. Source data are provided as Source Data files.