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. Author manuscript; available in PMC: 2016 May 21.
Published in final edited form as: Cell. 2015 May 7;161(5):1164–1174. doi: 10.1016/j.cell.2015.04.027

Figure 3. In vitro cleavage reflects in vivo targeting.

Figure 3

(A) Schematic of the substrate used to test for DNA cleavage in conditions where the crRNA matches the template strand. (B) In vitro DNA cleavage assay of the substrate show in panel A, with the radiolabel either in the template (left autoradiography) or non-template (right) strand. Reaction products were collected at 30, 60, 90 and 120 minutes. Reactions in which each of the components of the assay were omitted in a 120-minute assay are shown as controls. (C) Schematic of the “anti-tag” substrate in which the flanking sequence downstream on the nes target matches the 5' crRNA tag (light green), generating a full match between the crRNA and the DNA target. (D) In vitro DNA cleavage assay of the substrate show in panel C, with the non-template strand radiolabeled. Reaction products were collected at 30, 60, 90, 120 and 180 minutes. Reactions in which each of the components of the assay were omitted in a 120-minute assay are shown as controls.

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