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. 2021 Mar 8;49(6):3546–3556. doi: 10.1093/nar/gkab123

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — The HNH-domain of Cas9 catalyzes prespacer processing. (A) Schematic showing a list of complexes used to test prespacer processing by Urea-PAGE analysis. (B) Prespacer processing to a canonical size occurred only when prespacers were incubated with WT-supercomplex complexed with either a complementary or a noncomplementary sgRNA. Cas1-Cas2 integrase or supercomplex containing dCas9 did not show any prespacer processing. Quantification of the reactions shown have been represented as bar graphs. Error bars are representative of ±SD (n = 3). Time-point is 60 min. (C–E) Urea-PAGE gels showing kinetics of prespacer processing by supercomplex containing wild-type (WT)-Cas9 (C), RuvC-inactivated Cas9 (D10A-Cas9) (D) or HNH-inactivated Cas9 (H840A-Cas9) (E).