This PDF file includes:

• Fig. S1. DNA template design for single-molecule unzipping assay.
• Fig. S2. Characterization of the DNA elasticity parameters and the accuracy and precision of the unzipping method.
• Fig. S3. A representative unzipping trace of on-target dCas9 with two peaks.
• Fig. S4. Footprinting experiments with DNA bound by dCas9/sgRNA.
• Fig. S5. Mapping of dCas9/sgRNA-2/DNA and wtCas9/sgRNA-1/DNA interactions.
• Fig. S6. Forward unzipping with R15-20_mm and R11-20_mm sgRNAs.
• Fig. S7. EMSA confirmed the effect of the post-PAM DNA length on Cas9 binding.
• Fig. S8. DNA cleavage of wtCas9 with various DNA templates.
• Fig. S9. BLM unwinding through DNA/RNA hybrid chain.
• Fig. S10. Phi29 DNAP replicates through DNA-bound dCas9.
• Table S1. Sequences of sgRNAs and DNA.
• Table S2. Sequences of oligos used for bulk DNA cleavage assays.

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