Fig. 2: Gene expression programmed by the number of gRNA BS in synthetic operators.
From: A synthetic transcription platform for programmable gene expression in mammalian cells
A library of gRNA4, 7, and 10 synthetic operators containing 2×–16× gRNA BS was built to assess the number of gRNA BS that would be effective as a programmable parameter in controlling gene expression levels. Experimental groups (+), represented by red solid bars, were transiently transfected with four plasmids (P1–P4 in Fig. 1c) whereas gRNA-free control groups (−), represented by paired red hollow bars, were transfected without P1 (only P2–P4 in Fig. 1c) to detect operator leakage. EF1α and CMV promoters served as positive controls. Median mKate signals relative to the EF1α control were analyzed at 48 hours post-transfection with flow cytometry. a Synthetic operators of the gRNA4, 7, and 10 series with 2×–16× BS were examined in CHO-K1 cells (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(+) 2×, 4×, 6×, 8×, and 16× BS, gRNA7 P1(+) 2×, 4×, 8×, 12×, and 16× BS, and gRNA10 P1(+) 6×, 8×, 12×, and 16× BS vs. EF1α: p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0002, p = 0.0051, p < 0.0001, p = 0.0069, p = 0.0106, p = 0.0122, p < 0.0001, p < 0.0001, p = 0.0007, p < 0.0001, p < 0.0001, and p < 0.0001, respectively). b–e Synthetic operators of the gRNA10 series with 2×–16× BS were tested in b mouse C2C12 myoblasts (CMV and gRNA10 P1(+) 2×, 4×, 6×, 8×, 12×, and 16× BS vs. EF1α: p = 0.0016, p < 0.0001, p < 0.0001, p = 0.0018, p < 0.0001, p < 0.0001, p < 0.0001, respectively), c human HEK293T cells (CMV and gRNA10 P1(+) 6×, 8×, 12×, and 16× BS vs. EF1α: p < 0.0001, p = 0.0007, p < 0.0001, p < 0.0001, p < 0.0001, respectively), d rat H9C2 cardiomyoblast cells (gRNA10 P1(+) 6×, 8×, 12×, and 16× BS vs. EF1α: p = 0.0063, p < 0.0001, p < 0.0001, p < 0.0001, respectively), and e hiPSCs (gRNA10 P1(+) 8×, 12×, and 16× BS vs. EF1α: p = 0.0004, p < 0.0001, p < 0.0001, respectively). Data represent the mean ± SD (n = 3) (one-way ANOVA with multiple comparisons corrected by the Dunnett test). For increased expression (marked in black): *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001; for decreased expression (marked in blue): *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ND not detected. Source data are provided as Source Data files.
