Extended Data Figure 6: Design, purification, and DNA cleavage activity of ΔHNH-Cas9.
From: Conformational control of DNA target cleavage by CRISPR–Cas9
a, Domain organization of WT- and ΔHNH-Cas9 (top), showing the residues that were replaced with a GGS2 linker to generate ΔHNH-Cas9. Magnified view of connections between the HNH domain and RuvC II and III motifs in the apo (left) and sgRNA/DNA-bound (right) Cas9 crystal structures, as well as in the ΔHNH-Cas9 construct. Disordered linkers and the introduced GGS2 linker are shown as dashed lines. b, Size-exclusion chromatograms of WT- and ΔHNH-Cas9 using a Superdex 200 16/60 column (GE Healthcare). c, SDS–PAGE analysis of dCas9 (D10A/H840A), WT-Cas9, ΔHNH-Cas9, and the purified HNH domain (residues 776–907). Expected molecular masses are 159 kDa, 159 kDa, 142 kDa, and 16 kDa, respectively. For gel source data, see Supplementary Fig. 1. d, Representative radiolabelled DNA cleavage assay with WT-Cas9, ΔHNH-Cas9, ΔHNH-Cas9 in the presence of excess HNH domain, and HNH domain alone, resolved by denaturing PAGE.
