Target Specificity of Cas9 Nuclease via DNA Rearrangement Regulated by the REC2 Domain

Transcription

1 Supporting Information for: Target Specificity of Cas9 Nuclease via DNA Rearrangement Regulated by the REC2 Domain Keewon Sung, Jinho Park, Younggyu Kim, Nam Ki Lee, and Seong Keun Kim*, Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea. Department of Research and Development, LumiMac, Inc., Seoul 05805, Republic of Korea. * S1

2 Experimental methods Protein expression, purification, and dye labeling. All Cas9 variants were first subcloned into a pet- 28a(+) vector; DNA sequence encoding Cas9 from Streptococcus pyogenes with a nuclear localization signal, HA epitope, and 6 His-tag at the N-terminus was inserted. Mutations in the REC2 domain were introduced using Q5 Site-Directed Mutagenesis Kit (New England Biolabs) for deletion of residues N175- L302 and replacement with a GGSGGS linker in ΔREC2 Cas9, or using Gibson Assembly Master Mix (New England Biolabs or LumiMac) for multi-site-directed mutagenesis in REC2-#1~3 Cas9. Proteins were over-expressed in NiCo21(DE3) strain of E. coli (LumiMac) overnight at 18 C with 0.2 mm IPTG, and purified using Ni-NTA agarose resins (Qiagen). The eluate was further purified by size-exclusion chromatography on a Superdex 200 column (GE Healthcare) if necessary, and dialyzed against dialysis buffer (10 mm Tris-HCl ph 7.4, 300 mm NaCl, 0.1 mm EDTA, 1 mm DTT, and 10% (v/v) glycerol). Following dialysis, the Cas9 stock was concentrated using Amicon Ultra centrifugal filter-100 kda (Millipore) and ultra-centrifugated at 16,000 g for 10 min to remove protein aggregates. After the concentration and purity of Cas9 were quantified by the Bradford assay (Bio-Rad) and SDS-PAGE, respectively, the supernatant was diluted into Cas9 storage buffer composition (10 mm Tris-HCl ph 7.4, 300 mm NaCl, 0.1 mm EDTA, 1 mm DTT, and 50% (v/v) glycerol) for storage at -20 C without freezing. All the purification steps were performed at 4 C. To prepare Cas9 with dye-labeled REC2, C80L/C574E/D257C-mutated Cas9 was purified according to the above procedure. Purified Cas9 was again attached to the Ni-NTA resins and incubated with 3 equivalents of a maleimide-linked Cy5 derivative (LD650-MAL, Lumidyne Technologies) in labeling buffer (50 mm Tris-HCl ph 7.5, 300 mm NaCl, 5% (v/v) glycerol, 0.2 mm TCEP) overnight at 4 C with gentle mixing. Then, the Cas9-bound resins were re-loaded on a gravity flow column (Bio-Rad), and free dyes were removed by an excessive wash with the labeling buffer. Dye-labeled Cas9 was eluted by 250 mm imidazole added to the labeling buffer, followed by dialysis and centrifugation as described above; the concentration and labeling efficiency were checked with a UV/Vis spectrometer (Lambda 25, PerkinElmer). The final stock was stored in 10 mm Tris-HCl ph 7.4, 300 mm NaCl, 0.1 mm EDTA, 1 mm TCEP, and 50% (v/v) glycerol at -20 C. Nucleic acid preparation. All DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies. The NTS was biotinylated at the 5 end, followed by oligo-dt spacer to minimize side effects from surface immobilization. To construct dye-labeled DNA, amino-modified thymine was introduced into the DNA sequence, to which NHS ester-linked fluorophores were conjugated by amine- S2

3 NHS ester coupling reaction. For RNA labeling, Cy5-labeled crrna was directly purchased. The sequences for DNA and RNA constructs with the positions of biotin and amino-modified dt are listed in Table S1. Microscopy set-up and data processing. A prism-type total internal reflection fluorescence (TIRF) microscope was built for single-molecule fluorescence detection as previously described 1 with some modifications. A 532-nm laser (Samba, Cobolt AB) and a 638-nm diode laser (MLD, Cobolt AB) were used to excite Cy3 and Alexa 647, respectively. The fluorescence was collected by a water-immersion objective (UPlanApo 60, Olympus) with scattered light filtered out by an emission filter (ZET405/488/532/642m, Chroma Technology Corp.). The filtered signals were split into green and red channels by a dichroic mirror (645dcxr, Chroma Technology Corp.) and imaged simultaneously by a back-illuminated electron-multiplying charge-coupled device (ixon DU-897, Andor Technology). Dataacquisition time was set to 100 ms for FRET experiments and 200 ms for cleavage experiments. All images were recorded using a homemade C# script, followed by data processing using IDL and MATLAB codes. Pixelated fluorescence signals from each single-molecule spot were Gaussian-fitted to compute single-molecule fluorescence intensity. Then, FRET efficiency of the single molecule was calculated from IA /(γid + IA), where ID and IA are leakage- and background-corrected fluorescence intensities of the donor and acceptor, respectively, and the correction factor γ is defined as ΔIA /ΔID upon acceptor photo-bleaching. The value of γ was determined in the absence of proteins for the assay using Cy3-Alexa 647 dual-labeled DNA constructs (γ = 2 in our set-up), while in the presence of proteins for the labeling scheme of Cy3 at DNA and Cy5 at the protein (γ = 0.8 in our set-up). Single-molecule FRET assay. For all single-molecule imaging, quartz microscope slides and coverslips were passivated by polyethylene glycol to prevent non-specific binding of samples on the surface, and an imaging chamber was assembled with a surface-biotinylated quartz slide and coverslip according to the well-established protocol. 2 Biotinylated DNA constructs were then immobilized on the chamber surface via biotin-neutravidin interaction and imaged with or without Cas9:gRNA at 23 C in reaction buffer (unless otherwise specified): 50 mm Tris-HCl ph 7.9, 100 mm NaCl, 10 mm MgCl2, 1 mm DTT, 0.1 mg ml -1 BSA, and 5% (v/v) glycerol with an oxygen scavenging system (1 mg ml -1 glucose oxidase, 0.04 mg ml -1 catalase, and 0.8% (w/v) β-d-glucose) and a triplet quencher (~4 mm Trolox) for enhanced photostability. 3 Before imaging, 50 nm of grnas and 100 nm of Cas9 (20 nm of Cas9 for Cy5-labeled Cas9 to S3

4 reduce background signals) were pre-incubated for 10 min at 37 C to form Cas9:gRNA in the reaction buffer without the oxygen scavenger. After the injection of pre-incubated Cas9:gRNA mixed with the oxygen scavenger to the imaging chamber where DNA targets were immobilized, single-molecule movies were recorded successively for 30 min with the time window of ~2 min. From the data obtained after 20 min of incubation between DNA and Cas9:gRNA, the time trajectories that showed single-step photobleaching with both FRET donor and acceptor signals were manually selected to construct steady-state smfret histograms. In particular, for the histograms using dual-labeled DNA constructs, we further selected only the acceptor-bleached trajectories showing an abrupt increase in total intensity upon photobleaching. Since the γ factor was determined in the absence of Cas9:gRNA, the mismatch of the total intensity upon the acceptor-bleaching is likely caused by Cas9:gRNA binding (i.e., PIFE); thus, this additional criterion enables us to sort out only the trajectories of Cas9:gRNA-bound molecules. From the selected trajectories, the first 5-s FRET data were extracted to build the histograms of the FRET efficiency upon binding; the data from over 200 molecules were employed to build each histogram. The relative population of each FRET state was calculated from a double-gaussian fit of the histogram. For dwell-time analysis, fragments of the time trajectories that show at least one FRET transition after the initial transition to the low-fret state were collected from all data recorded during the 30 min of imaging time. These raw smfret trajectories were fitted into two states, applying the variational Bayesian method in vbfret, 4 to measure the dwell time during repetitive transitions in each FRET state. For experiments to capture the initial FRET transition right after the binding of Cas9:gRNA to DNA, the concentration of Cas9:gRNA (10 nm grnas, 20 nm Cas9) was lowered to observe a higher number of binding events for an extended time period. The dwell time between binding and initial displacement was manually measured for each single-molecule time trajectory containing the binding moment. Single-molecule cleavage assay. To quantify Cas9-mediated cleavage of DNA, we calculated the ratio between the number of Cy3 and Alexa 647 molecules on DNA before and after the incubation with Cas9:gRNA as previously described. 1 Unlike our previous report, we employed green-red laser alternation to determine the ratio between the number of Cy3-Alexa 647 labeled DNA molecules and the number of all Cy3-labeled DNA molecules since FRET occurs between the two fluorescent dyes (Figure S1). As for the single-molecule FRET assay, 50 nm of grnas and 100 nm of Cas9 (unless described otherwise) were pre-incubated for 10 min at 37 C to form Cas9:gRNA in the reaction buffer without the oxygen scavenger. Then, the cleavage reaction was performed by incubating Cas9:gRNA with immobilized DNA in the single-molecule imaging chamber at room temperature in the reaction-buffer condition without the oxygen scavenger. After 1 hr of incubation (unless otherwise stated), the reaction was quenched, and the S4

5 Alexa 647-labeled cleaved fragments were dissociated by the injection of 7M-urea solution, followed by a rapid wash to prevent denaturation of the DNA duplex. The ratiometric comparison excluded errors from the urea-induced disruption of biotin-neutravidin interaction, and errors from photo-bleaching were negligible due to the short imaging time (~4 s). The data from more than 4,000 molecules were used to determine the cleaved fraction of DNA for individual experiments. DNA binding assay. A bulk protein-induced fluorescence enhancement (PIFE) technique was used to measure DNA-binding affinities of various Cas9 species. 10 nm dual-labeled DNA was incubated at 37 ºC for 2 hours in binding buffer (50 mm Tris-HCl ph 8.0, 100 mm NaCl, 5 mm EDTA, 1 mm DTT, and 5% (v/v) glycerol) with increasing concentrations of Cas9:gRNA (0, 0.1, 1, 3, 10, 30, 100, 300, 800 nm). Following the incubation, PIFE (%), defined as 100 (%) where I and I are minimum and maximum fluorescence intensity respectively, was calculated by measuring the total fluorescence intensity (I) in the range of nm upon 532-nm excitation using a fluorometer (QM- 4/2005SE, Photon Technology) to quantify the fluorescence enhancement upon Cas9:gRNA binding. 5 All experiments were repeated at least twice. S5

6 Figure S1. Surface-immobilized dual dye-labeled DNA is well cleaved by Cas9:gRNA. (a) CCD fluorescence images and schematic structures of on-target DNA labeled with Cy3 and Alexa 647 before and after the cleavage reaction by Cas9:gRNA. To detach the cleaved DNA fragment containing the Alexa 647 label, 7 M-urea solution was injected (refer to the single-molecule cleavage assay in the experimental methods section for detail). (b) To verify the cleavage activity of Cas9:gRNA toward the surfaceimmobilized DNA with dual labels, Cas9-induced DNA cleavage reaction was monitored in time- and concentration-dependent manner using the single-molecule cleavage assay. (c) As negative controls, we employed dcas9 (catalytically inactivated mutant Cas9) and WT Cas9 without Mg 2+ ion (reaction buffer with no MgCl2 and 5 mm EDTA). The measured fraction of cleaved DNA that includes urea-mediated denaturation of dsdna was negligible in both cases as in the case of DNA-only incubation; mean and s.e.m. (n = 2 or 3) are shown. S6

7 Figure S2. Cy5-labeled crrna enables direct identification of Cas9:gRNA-DNA binding. As a control, Cy5-crRNA was newly employed to directly monitor the binding moment, in which the Cy5 dye was labeled at the 3 end of crrna far removed from the Cy3 position on the NTS within Cas9:gRNA:DNA. (a) Schematic diagrams (left) and FRET histograms (right) for Cy3-labeled on-target DNA (upper) and Cy3-Alexa 647 dual-labeled on-target DNA (lower) constructs bound to the Cy5- crrna-containing Cas9 complex (Cas9:Cy5-gRNA); we combined our in-house alternating laser excitation (ALEX) technique with TIRF microscopy to discriminate Cas9:Cy5-gRNA-bound DNA molecules. The upper histogram shows that no FRET occurs between Cy3 on the NTS and Cy5 on crrna after the binding, while the lower histogram shows that the Cy5-labeled Cas9:gRNA results in nearly identical FRET states with similar relative population compared to non-labeled Cas9:gRNA (Figure 1b). S7

8 (b) A representative time trajectory recorded with ALEX right after the injection of Cas9:Cy5-gRNA to dual-labeled on-target DNA. When the FRET efficiency increases from the DNA-only to the high-fret value with the abrupt enhancement of total intensity (PIFE) upon green-laser excitation (top and middle), intensity of the red fluorophores (Alexa 647 and Cy5) upon red-laser excitation synchronously is doubled (bottom), which directly indicates binding of Cas9:Cy5-gRNA. Among 167 single-molecule trajectories showing a 2-fold increase in the red-excitation intensity, virtually all molecules (164/167; 98%) exhibit a synchronous PIFE and FRET transition, which confirms that these are indeed caused by the binding of Cas9:gRNA. S8

9 Figure S3. Catalytically inactivated (D10A/H840A) dead Cas9 (dcas9) yields nearly identical sub-conformational dynamics for the NTS. (a) A steady-state FRET histogram (upper) and representative single-molecule time trajectory (lower) for on-target DNA with dcas9. (b) Fractions of Cas9-bound DNA molecules showing time trajectories stably docked at the high-fret state (red), transitioning between the two FRET states (yellow), and stably docked at the low-fret state (green). For on-target DNA interacting with WT Cas9, only 8% of Cas9-bound DNA molecules exhibit time trajectories with dynamic transitions (left). However, when we replace WT Cas9 to dcas9, the dynamic fraction increases sharply to 34% (right), indicating that the dynamic behavior occurs mostly within the non-cleaved Cas9:gRNA:DNA complex rather than the cleaved product. The total numbers of molecules are 789 and 658 for WT Cas9 and dcas9, respectively. S9

10 Figure S4. A higher degree of PAM-distal mismatch shifts the dynamic equilibrium from the low- FRET to high-fret state and reduces the cleavage efficiency of Cas9 nuclease. (a) Representative single-molecule time trajectories for on-target DNA and three off-target DNAs with PAM-distal mismatches (M19-20, M18-20, and M17-20). Mean FRET values ± s.e.m. (n = 3) of the two FRET states obtained by double-gaussian fitting of the FRET histograms in Figures 1b and d are shown. To note, the low-fret value shifts down as the number of mismatches increases from 0- to 3-bp, while the high- FRET value remains constant, indicating that the structure of the low-fret state is affected by the PAMdistal mismatch. (b) Fraction of the low-fret population and Cas9 cleavage efficiency for the four DNA constructs (mean ± s.e.m., n = 3). S10

11 Figure S5. NTS displacement occurs regardless of Mg 2+ ion although a divalent cation is essential for rearrangement of the HNH nuclease domain and cleavage reaction. Steady-state FRET histograms for on-target DNA and three off-target DNAs with PAM-distal mismatches (M19-20, M18-20, and M17-20) upon interacting with WT Cas9 in Mg 2+ -free condition (reaction buffer with no MgCl2 and 5 mm EDTA); FRET efficiencies of the two FRET peaks were changed compared to the Mg 2+ -containing condition (Figure 1) due to difference in ionic strength. This result shows that the D conformation forms even when HNH remains at its inactive state ( conformational checkpoint ), 6 indicating that the NTS and HNH do not move in a synchronous manner. Since the HNH rearrangement toward its active state in the presence of Mg 2+ requires the NTS displacement 6,7 while the NTS displacement does not need the HNH activation as shown above, it is likely that in the cleavage reaction pathway, the NTS is first displaced S11

12 toward the D conformation followed by the HNH transition toward its active structure that further stabilizes the D conformation by interacting with the displaced NTS. 7,8 Correspondingly, the fraction of the D conformation of the NTS for on-target DNA and off-target DNAs with the PAM-distal mismatches is larger for the Mg 2+ -containing condition (Figure 1) than for this Mg 2+ -free condition. S12

13 Figure S6. Comparison between an available crystal structure and the measured FRET efficiencies of the NTS sub-conformations suggests the REC2 domain as a putative mediator for the NTS dynamics. The crystal structure of Cas9:gRNA:DNA (PDB ID: 5F9R) 8 in surface view showing REC2 (pink), NTS (magenta), guide RNA (beige), TS (navy), and RuvC (light blue), with HNH omitted for clarity. The RuvC residues interact with the PAM-proximal region of the NTS in the D conformation. 8,9 However, in order to explain the regulation of the NTS dynamics in response to the PAM-distal basecomplementarity (Figure 1d), we hypothesized that a separate Cas9 domain may get involved prior to the RuvC docking of the NTS in the D conformation (i.e., in the I conformation). (a) The FRET efficiency of the D conformation for on-target DNA (0.44) amounts to 53 Å using the Förster radius of 51 Å, 10 which is consistent with the inter-dye distance of 52 Å in reference to the crystal structure (yellow dotted line connecting the green and red circles). (b) The FRET efficiency of the I conformation (0.74) amounts to an inter-dye distance of 43 Å; thus, the Cas9 residues Å apart from the Alexa 647-labeled position S13

14 (red circle) are highlighted in green to cover the possible location of the Cy3-labeled NTS in the I conformation. According to our previous study, the PAM-proximal heteroduplex (~10-bp long) between crrna and the TS of DNA is formed immediately upon Cas9 binding. 1 Given this knowledge, it is likely that, in the I conformation, at least the PAM-proximal 10-nt segment of the NTS containing the Cy3 label is in the form of flexible ssdna that is unwound from the TS but still remains close to it (Figure 1d, cartoon at the top-right corner). Among the domains within the green sphere centered at the Alexa 647- labeled site, the REC2 domain also lies near the PAM-proximal to PAM-mid region of the TS within the heteroduplex. Thus, we speculated that the rearrangement of the PAM proximal-to-mid region of the NTS would be mediated by REC2. S14

15 Figure S7. Cas9 labeled at the D257C residue on REC2 with Cy5 shows that the NTS approaches REC2 in the I conformation. (a) Cy3 and Cy5 labeling positions respectively represented by green and red circles, in reference to the crystal structure of Cas9:gRNA:DNA (PDB ID: 5F9R) 8 that shows REC2 (pink), NTS (magenta), guide RNA (beige), TS (navy), and RuvC (light blue), with HNH omitted for clarity. (b) Comparison between the cleavage kinetics of 20 nm WT Cas9:gRNA (blue) and Cy5- Cas9:gRNA (red) for on-target DNA shows that the C80L/C574E/D257C mutation and dye labeling do not significantly impair the activity of Cas9 nuclease. (c) Schematic diagram of smfret assay using Cy3-labeled DNA and Cas9 labeled by Cy5 on the REC2 surface (upper panel) with the same colorcoding scheme as in a. FRET histograms for on-target DNA and three off-target DNAs with PAM-distal mismatches (M19-20, M18-20, and M17-20) using Cy3-DNA and Cy5-REC2 Cas9 (lower panel). Contrary to Figure 1, the low-fret value now shifts up slightly as the number of mismatches increases, while the high-fret value remains constant as in Figure 1; Elow = 0.30 ± , 0.33 ± , 0.35 ± 0.005, and 0.36 ± 0.06 and Ehigh = 0.75 ± 0.009, 0.76 ± 0.002, 0.75 ± 0.002, and 0.76 ± respectively for on-target, M19-20, M18-20, and M17-20 DNA. S15

16 Figure S8. REC2-deleted Cas9 (ΔREC2 Cas9) reduces relative population in the I conformation. (a) Schematic of domain organization for ΔREC2 Cas9. (b) The cleavage kinetics of 50 nm ΔREC2 Cas9:gRNA (magenta) and WT Cas9:gRNA (blue) for on-target DNA. (c) FRET histograms for on-target DNA and three off-target DNAs with PAM-distal mismatches (M19-20, M18-20, and M17-20) using ΔREC2 Cas9. S16

17 Figure S9. Cleavage kinetics and DNA-binding affinities of REC2-charge-mutated Cas9 species. (a) Cleavage kinetics of WT and three REC2-charge-mutated Cas9 species toward on-target DNA. (b) DNAbinding affinities of WT and three REC2-charge-mutated Cas9 species with on-target DNA. Error bars represent s.e.m. (n = 2 or 3). S17

18 Figure S10. smfret data for REC2-#1 Cas9. (a) FRET histograms for on-target DNA and three offtarget DNAs with PAM-distal mismatches (M19-20, M18-20, and M17-20) interacting with a REC2- charge-mutated species (REC2-#1 Cas9). (b) A representative time trajectory for M19-20 DNA with REC2-#1 Cas9. The dwell time between binding and initial displacement ( t ), the D-conformational dwell time ( t(e )), and the I-conformational dwell time during the subsequent repetitive transitions ( t(e )) are illustrated. (c) Distributions of the dwell time between binding and initial displacement of on-target DNA and three off-target DNAs with the PAM-distal mismatches when interacting with WT Cas9 (blue) and REC2-#1 Cas9 (red) fitted to single-exponential curves (black); the fitting results are shown at a mean ± s.d. S18

19 Figure S10 (continued). (d) Dwell time distributions for the repetitive transitions between the I and D conformations with bi-exponential (black, solid) vs single-exponential (dotted) fits; both I and D conformations are composed of short-lived (major component) and long-lived (minor component) states. Given the similar repetitive transitioning behavior of HNH, 6 it is likely that the two components in each NTS conformation refer to different HNH-conformational states (described in detail below). In each plot, S19

20 the amplitude-weighted average dwell time ( t ) calculated by the equation at the top is shown at a mean ± s.d. (all fitting results are listed in Table S2). For the on-target DNA interacting with REC2-#1 Cas9, we did not analyze the transitional dwell times since sub-conformational transitions were rarely observed. (e) The dwell times in the long-lived state of the repetitive-i conformation (blue and red bars) well match with those in the initial-i conformation, i.e., dwell time between binding and initial displacement, (white bars; same as in Figure 2c) for both WT (left panel) and REC2-#1 Cas9 (middle panel). This indicates that the long-lived state corresponds to the initial-i conformation in which HNH is placed far apart from its catalytically active position and from REC2 ( RNA-bound state ), 6,11 thereby allowing full access of the NTS to the REC2 surface. Considering that HNH mainly undergoes the repetitive transitions between the active state and the other inactive state ( conformational checkpoint ), 6 the major short-lived component of the repetitive-i conformation would be the I conformation of the NTS with HNH at its checkpoint. A structural study indicates that the checkpoint structure of HNH partially blocks the positively charged REC2 surface, 12 and correspondingly the dwell time in the short-lived state depends less on the REC2-charge neutralization compared to the case of the long-lived state (Table S2). On the other hand, the two kinetic states of the D conformation are likely to represent the active and checkpoint states of HNH as described in the caption of Figure S5. Relative amplitude of the long-lived state of the D conformation shows sharp decrease along with increase in PAM-distal mismatches for both WT (blue) and REC2-#1 Cas9 (red) (right panel), which suggests that the long-lived state would be the D conformation of the NTS with HNH in its catalytically active state. For the D-conformational dwell time, both kinetic states show increases in the dwell times upon the REC2 mutation (Table S2), indicative of allosteric coupling between REC2 and the displaced NTS. S20

21 Figure S11. smfret data for REC2-#2 and REC2-#3 Cas9. (a) FRET histograms for on-target DNA and three off-target DNAs with PAM-distal mismatches (M19-20, M18-20, and M17-20) interacting with REC2-#2 (yellow) and REC2-#3 Cas9 (green). (b and c) Kinetic results for M18-20 DNA with various Cas9 species (mean ± s.d.). The REC2-#2 and -#3 Cas9 show similar effects on the steady-state population and kinetics between the NTS conformations compared to REC2-#1 except for the dwell time between binding and initial displacement (i.e., dwell time in the initial-i conformation). S21

22 Figure S12. Cleavage efficiencies of WT and REC2-charge-mutated Cas9 species toward off-target DNAs with PAM-distal mismatches. Mean and s.e.m. (n = 2 or 3) are shown. S22

23 Table S1. Sequence list for DNA and RNA constructs. iammc6t refers to amino-modified dt for dye labeling, and the mismatched bases in off-target sequences are colored in red. Description Type Sequences On-target DNA non-target strand target strand M19-20 DNA M18-20 DNA M17-20 DNA M18-19 DNA M17-18 DNA M16-17 DNA M15-16 DNA crrna tracrrna Cy5-crRNA non-target strand target strand non-target strand target strand non-target strand target strand non-target strand target strand non-target strand target strand non-target strand target strand non-target strand target strand ssrna ssrna ssrna 5 [Biotin] ttt ttt GAG GAA GTG CCT GAG TCC GAG C/iAmMC6T/G AAG AAG AAA GGC ACA CAT CAC ATC 3 5 GAT GTG ATG TGT GCC /iammc6t/tt CTT CTT CAG CTC GGA CTC AGG CAC TTC CTC 3 5 [Biotin] ttt ttt GAG GAA GTG CCT CTG TCC GAG C/iAmMC6T/G AAG AAG AAA GGC ACA CAT CAC ATC 3 5 GAT GTG ATG TGT GCC /iammc6t/tt CTT CTT CAG CTC GGA CAG AGG CAC TTC CTC 3 5 [Biotin] ttt ttt GAG GAA GTG CCT CTC TCC GAG C/iAmMC6T/G AAG AAG AAA GGC ACA CAT CAC ATC 3 5 GAT GTG ATG TGT GCC /iammc6t/tt CTT CTT CAG CTC GGA GAG AGG CAC TTC CTC 3 5 [Biotin] ttt ttt GAG GAA GTG CCT CTC ACC GAG C/iAmMC6T/G AAG AAG AAA GGC ACA CAT CAC ATC 3 5 GAT GTG ATG TGT GCC /iammc6t/tt CTT CTT CAG CTC GGT GAG AGG CAC TTC CTC 3 5 [Biotin] ttt ttt GAG GAA GTG CCT GTC TCC GAG C/iAmMC6T/G AAG AAG AAA GGC ACA CAT CAC ATC 3 5 GAT GTG ATG TGT GCC /iammc6t/tt CTT CTT CAG CTC GGA GAC AGG CAC TTC CTC 3 5 [Biotin] ttt ttt GAG GAA GTG CCT GAC ACC GAG C/iAmMC6T/G AAG AAG AAA GGC ACA CAT CAC ATC 3 5 GAT GTG ATG TGT GCC /iammc6t/tt CTT CTT CAG CTC GGT GTC AGG CAC TTC CTC 3 5 [Biotin] ttt ttt GAG GAA GTG CCT GAG AGC GAG C/iAmMC6T/G AAG AAG AAA GGC ACA CAT CAC ATC 3 5 GAT GTG ATG TGT GCC /iammc6t/tt CTT CTT CAG CTC GCT CTC AGG CAC TTC CTC 3 5 [Biotin] ttt ttt GAG GAA GTG CCT GAG TGG GAG C/iAmMC6T/G AAG AAG AAA GGC ACA CAT CAC ATC 3 5 GAT GTG ATG TGT GCC /iammc6t/tt CTT CTT CAG CTC CCA CTC AGG CAC TTC CTC 3 5 GAG UCC GAG CUG AAG AAG AAG UUU UAG AGC UAU GCU GUU UUG 3 5 AGC AUA GCA AGU UAA AAU AAG GCU AGU CCG UUA UCA ACU UGA AAA AGU GGC ACC GAG UCG GUG CUU U 3 5 GAG UCC GAG CUG AAG AAG AAG UUU UAG AGC UAU GCU GUU U/Cy5/ 3 S23

24 Table S2. Bi-exponential fitting result for the dwell-time analysis in Figure S10d. I and D refer to the I and D conformation, respectively. The first component (A, t ) corresponds to the long-lived one, and the second (A, t ) to the short-lived component (A: normalized amplitude, t: time constant). The fitting results are shown at a mean ± s.d. On-target M19-20 M18-20 M17-20 WT Cas9 REC2-#1 Cas9 I D I D A 0.18 ± ± ± ± 0.02 t 1.82 ± 0.23 (s) 2.07 ± 0.11 (s) 2.87 ± 0.19 (s) 6.64 ± 1.52 (s) A 0.82 ± ± ± ± 0.03 t 0.42 ± 0.02 (s) 0.48 ± 0.01 (s) 0.59 ± 0.01 (s) 1.16 ± 0.04 (s) A 0.38 ± ± ± ± 0.01 t 2.87 ± 0.26 (s) 2.61 ± 0.19 (s) 2.80 ± 0.17 (s) 1.58 ± 0.11 (s) A 0.62 ± ± ± ± 0.01 t 0.50 ± 0.06 (s) 0.54 ± 0.03 (s) 0.45 ± 0.01 (s) 0.28 ± 0.01 (s) A 0.17 ± ± ± 0.08 t 2.10 ± 0.11 (s) 2.47 ± 0.11 (s) 2.75 ± 0.41 (s) A 0.83 ± ± ± 0.11 t 0.26 ± 0.01 (s) 0.50 ± 0.01 (s) 0.90 ± 0.08 (s) A 0.32 ± ± ± 0.03 t 3.80 ± 0.31 (s) 4.34 ± 0.30 (s) 2.81 ± 4.63 (s) A 0.68 ± ± ± 0.05 t 0.92 ± 0.06 (s) 0.80 ± 0.03 (s) 0.45 ± 0.02 (s) S24

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