Extended Data Fig. 7: The formation of an 18-nt guide-target DNA duplex is essential for the complete activation of Cas9.
From: Trans-nuclease activity of Cas9 activated by DNA or RNA target binding
a-c, (Top) Fluorescence time-course assays showing the sensitivity of SpyCas9-catalyzed trans-cleavage using 13-nt (a), 11-nt (b), and 8-nt (c) ssDNA-FQ substrates and increasing target activator concentrations. (Bottom) Comparison of slopes of curves within 10-25 min from the top time-course graphs. The slope was calculated by simple linear regression. Data are shown as mean ± s.d. (n = 3 independent experiments). Statistical analysis was by two-tailed t test. d, (Top) Comparison of trans-cleavage rates within 10-20 min for SpyCas9 using dsDNA activators with indicated mismatches. (Bottom) Sequences of dsDNA activators and the complementary guide RNAs used in reactions. Data are shown as mean ± s.d. (n = 3 independent experiments). e-f, Time-course assays (e) and maximum fluorescence signals (f) showing that the DNA target length affects the trans-cleavage activity of SpyCas9. Data are shown as mean ± s.d. (n = 3 independent experiments).
