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. Author manuscript; available in PMC: 2018 Apr 27.
Published in final edited form as: Nature. 2017 Sep 20;550(7676):407–410. doi: 10.1038/nature24268

Figure 2. The alpha-helical lobe regulates HNH domain activation.

Figure 2

a, Domain organization of SpCas9ΔREC3. b, On-target DNA cleavage assay using SpCas9ΔREC3 with increasing concentrations of the REC3 domain supplied in trans, resolved by denaturing PAGE; repeated three independent times with similar results. c, Schematic of SpCas9ΔREC3HNH, with REC3 added in trans. Inactive to active structures represent HNH in the sgRNA-bound (PDB ID: 4ZT0) to dsDNA-bound (PDB ID: 5F9R) forms, respectively. d, smFRET histograms showing HNH conformation with SpCas9ΔREC3HNH bound to an on-target substrate, with and without REC3. e, Schematic of SpCas9REC2; HNH domain is omitted for clarity. Inactive to active structures represent REC2 in the sgRNA- (PDB ID: 4ZT0) to dsDNA-bound (PDB ID: 5F9R) forms, respectively. f–g, smFRET histograms showing REC2 conformation with f, WT SpCas9REC2 and g, SpCas9-HF1REC2 bound to on-target and mismatched targets. For panels d, f and g, black curves represent a fit to multiple Gaussian peaks.