Preparation of guide-RNA and Cas9-RNA.

Guide-RNA for Cas9 is a combination of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). For smFRET assay for DNA interrogation by Cas9-RNA, crRNA with an amino-dT was purchased from IDT and labeled with Cy5-NHS. Location of the Cy5 in the crRNA is shown in Supplementary Figure 1 The tracrRNA was in vitro transcribed as described previously⁴. For DNA unwinding smFRET assay, both crRNA and tracrRNA were unlabeled and transcribed in vitro. Guide-RNA was assembled by mixing crRNA and tracrRNA at 1:1.2 ratio in buffer containing 10 mM Tris HCl (pH 8) and 50 mM NaCl, heated to 90 °C and slowly cooled to room temperature. Cas9-RNA was assembled by mixing guide-RNA and Cas9 at a ratio of 1:3 in Cas9-RNA activity buffer (20 mM Tris HCl (pH 8), 100 mM KCl, 5 mM MgCl₂, 5% v/v glycerol). Cas9-RNA cleavage activity on cognate sequence used for smFRET assay for DNA interrogation was characterized previously⁴. We used a slightly difference sequence for optical placement of amino-dTs for DNA unwinding smFRET assay and Cas9RNA cleavage activity on that sequence was also confirmed (Supplementary Fig. 4). RNA sequences are available in Supplementary Table 1 & 2. For smFRET assay of DNA unwinding by surface-tethered Cas9-RNA, a biotin adaptor DNA strand was annealed to a complementary extension on guide-RNA (Supplementary Figure 7).