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. 2022 Mar 2;603(7900):343–347. doi: 10.1038/s41586-022-04470-1

Fig. 2. Positions 12–14 of the gRNA–TS duplex occupy a blind spot for REC3 mismatch detection.

Fig. 2

a, b, Structures of 12–14 MM at 5 min (a) and 1 h (b) in linear and kinked conformations, respectively. The position of the 12–14 MM is shown as light green and light pink for the gRNA and the target strand, respectively. Models are shown as isosurface representations. c, Conformational change of the PAM-distal gRNA–TS duplex. The Cas9 protein structure is largely unchanged (root-mean-square deviation (RMSD) of less than 2 Å for equivalent C-alpha atoms), but the PAM-distal gRNA–TS duplex end undergoes a 30 Å conformational change, docking with REC3. d, Close-up view of positions 12–14, showing that because of the phase of the gRNA–TS duplex, REC3 makes no contacts with these base pairs. e, Schematic of interactions between REC3 and positions 9–17 of the gRNA–TS duplex. No interactions occur between Cas9 REC3 and positions 12–14 MM. Position 1 of the duplex is the first base of the target strand that hybridizes with the gRNA spacer.