Science Advances

Supplementary Materials

This PDF file includes:

  • fig. S1. Steady-state smFRET measurements and bulk cleavage assays of Cas9 labeled with Cy3/Cy5 FRET pairs.
  • fig. S2. Representative smFRET trajectories reveal three different stable conformations of Cas9 under saturating DNA concentrations.
  • fig. S3. Steady-state smFRET histograms of a reciprocal Cas9 variant.
  • fig. S4. DNA immobilization of Cas9-sgRNA to the PEG surface eliminates the complexes that are unable to bind the DNA.
  • fig. S5. Dwell time analysis of dynamic transitions of Cas9 in the absence and presence of the DNA substrates.
  • fig. S6. Specific binding of Cas9 to the on-target DNA substrate immobilized to the PEG surface.
  • fig. S7. Conformational dynamics of the HNH domain observed at 2 Hz.
  • fig. S8. DNA cleavage activity of Cas9 in the presence of various divalent cations.
  • fig. S9. Conformational dynamics of the HNH domain in the presence of 10 μM Mg2+.
  • fig. S10. smFRET histograms of dCas9 bound to on-target dsDNA in the absence and presence of a divalent cation.
  • table S1. List of RNA and DNA substrates used in this study.
  • table S2. Expected and measured EFRET values for Cas9HNH-1 and Cas9HNH-2.
  • Legends for movies S1 and S2
  • Reference (32)

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Other Supplementary Material for this manuscript includes the following:

  • movie S1 (.avi format). Real-time cleavage of an on-target pdDNA1 by Cas9.
  • movie S2 (.avi format). Real-time cleavage of a 1–3 bp mm pdDNA1 by Cas9.

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