Supplementary Materials

Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage

Fuguo Jiang, David W. Taylor, Janice S. Chen, Jack E. Kornfeld, Kaihong Zhou, Aubri J. Thompson, Eva Nogales, Jennifer A. Doudna

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S11
  • Tables S1 and S2
  • References

Images, Video, and Other Other Media

Movie S1
Architecture of the sgRNA-bound Cas9. This movie shows 360° rotations of the sgRNA-bound Cas9 reconstruction with and without the crystal structure (PDB 4ZT0) (14) docked into the structure. First, the sgRNA is shown with the 5'-most ten nucleotides (nts. 1–10) of the guide region of the RNA built into the EM density. Second, the Hel I–III, Arg and CTD domains are added; third, the RuvC and HNH domains are added to show the architecture of the entire complex. The crystal structure for sgRNA-bound Cas9 is docked as in Fig. S10B.
Movie S2
Architecture of the dsDNA target-bound Cas9. This movie shows 360° rotations of the DNA-bound Cas9 reconstruction with and without the crystal structure (this study) docked into the structure. First, the sgRNA is shown, excluding the 20 nts of the guide region; second, the sgRNA:target-strand DNA heteroduplex, PAM duplex, non-target DNA strand and PAM-distal duplex are added; third, the Hel I–III, Arg and CTD domains are added; fourth the RuvC and HNH domains are added to show the architecture of the entire complex. The crystal structure for DNA-bound Cas9 is docked as in Fig. S10D.
Movie S3
Conformational rearrangements of RNA-bound Cas9 upon target-DNA binding and R-loop formation. This movie shows a morph between the RNA-bound Cas9 and DNA-bound Cas9. First, RNA-bound Cas9 is shown. Next, to emphasize the conformational rearrangements required for binding target DNA and formation of an R-loop, the domains of Cas9 from the RNA-bound structure are morphed from their RNA- to DNA-bound position. Once in the DNA-bound state, the sgRNA:target-strand DNA heteroduplex, PAM duplex, non-target DNA strand and PAM-distal DNA duplex density are shown in medium blue. The movie then repeats in reverse (from DNA-bound to the RNA-bound state). The morph was generated using the morph map function in UCSF Chimera. The two maps were aligned based on the sgRNA density, and morph maps were generated for each individual domain.