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The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation

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Science Advances  13 Nov 2019:
Vol. 5, no. 11, eaaw9807
DOI: 10.1126/sciadv.aaw9807
  • Fig. 1 Experimental configuration for single-molecule unzipping experiments and dCas9/sgRNA/DNA interaction mapping.

    (A) Cartoon illustrating the single-molecule DNA unzipping experiment used for the detection of the interactions between dCas9/sgRNA and DNA. The sequence of the steps of the unzipping of the DNA template is numbered as illustrated. (B and E) Representative traces of forward (black) and reverse unzipping (blue) in the presence of dCas9 showing the force versus number of base pairs unzipped. The naked DNA unzipping signatures are also presented for comparison (gray). The light-yellow rectangle shows the expected Cas9 binding position. Red arrows indicate the force peaks. Insets: Zoom in of the regions with increases in force. (C and F) Histogram of the disruption forces of dCas9/sgRNA. The data and their Gaussian fits are shown. (D and G) Histogram of the positions of dCas9/sgRNA and target DNA interactions along the DNA sequence. The data and their Gaussian fits are shown. The black and blue triangles indicate the pre- and post-PAM interactions, respectively.

  • Fig. 2 Interactions of dCas9/sgRNA with a PAM-distal mismatched DNA target.

    (A to F) Representative traces of forward and reverse DNA unzipping with the D15-20mm (A, B, and C) and the D11-20mm templates (D, E, and F). The forward and reverse unzipping forces versus the number of base pairs are presented in black and blue, respectively. The naked DNA unzipping signatures are also presented for comparison (gray). Red arrows indicate the force peaks. The forward unzipping forces populated two signatures: one peak and dual peaks. (G) Positions and forces of the three interactions between Cas9/sgRNA and mismatched DNA: pre-, post-, and intermittent interaction. The hollow triangle indicates the transient interaction, and the black and blue triangles indicate the pre- and post-PAM interactions, respectively. (H) Fractions of dCas9-bound DNA at the expected position with fully matched (target) and mismatched DNAs (D15-20mm and D11-20mm), as revealed by forward unzipping.

  • Fig. 3 Post-PAM interaction is essential for Cas9 binding and cleavage.

    (A) Schematic diagram of the unzipping of DNA templates with various DNA lengths downstream of the PAM (represented by X). Representative traces of forward unzipping of the “Post-30” DNA templates. (B) Fraction of dCas9-bound DNA as a function of dCas9 concentration. DNA templates with various DNA lengths are shown in different colors. (C) A representative gel showing Cas9 cleavage of DNA templates with various DNA lengths downstream of the PAM. Reactions were quenched at five time points (0, 5, 10, 15, and 30 min). (D) Schematic diagram of the Cas9 sequential binding assay. Representative traces of forward unzipping of the PAM-in DNA template with 30-bp dsDNA remaining between the two PAMs show the force versus the number of base pairs unzipped. These traces were obtained when both the 1st and the 2nd dCas9 were present. The naked DNA unzipping signatures are also presented for comparison (gray). (E) Fraction of the second dCas9-bound DNA in the presence and absence of the 1st dCas9. N represents the number of bases between the two dCas9 exclusive PAM regions. (F) A representative gel showing Cas9 cleavage of a PAM-in DNA template with 10 bp of separation between the two PAMs. Reactions were quenched at five time points (0, 1, 2, 3, and 5 min).

  • Fig. 4 BLM unwinds through DNA-bound dCas9.

    (A and C) The BLM unwinding was initiated from either the upstream (A) or downstream (C) side of the PAM. Representative traces showed the number of unwound base pairs versus time under an assisting force of 12 pN in the presence or absence of prebound dCas9. For clarity, the traces have been shifted along the time axis. The dashed lines indicate the expected dCas9 binding position. (B and D) Histogram showing the pause time of BLM at the expected dCas9 binding position when unwinding started on the upstream (B) or downstream (D) side of the PAM. These time distributions followed a single exponential distribution with average times of 51 and 17 s, respectively.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaaw9807/DC1

    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 R15-20mm and R11-20mm 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.

  • Supplementary Materials

    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 R15-20mm and R11-20mm 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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