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. 2019 Nov 13;5(11):eaaw9807. doi: 10.1126/sciadv.aaw9807

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Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

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Fig. 3 — (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).