Fig. 3: R-loop formation directs the non-target strand toward the HD domain.

a R-loop formation begins with unwinding of the PAM-proximal duplex with the target strand (cyan) traveling up along the Cas7 filament and the non-target strand traveling across the front of Cas10d. b Electrostatic surface potential and conservation scores appear correlated, where positive patches are more highly conserved and coordinate the non-target strand towards the active site on the back of Cas10d. Dashed line (black) represents the putative path of the NTS towards the active site of Cas10d, where nicking occurs. c Active site of Cas10d is highly conserved relative to the rest of the Cas10d surface. d H115, D116, H81, D210, and H214 form the active site of Cas10d to cleave the non-target strand. These four residues are highly conserved and likely coordinate a divalent cation. e Crystal structure of AcrID1 (PDB: 6thh, (Manav et al.²⁸)) overlayed onto the Cas10d subunit of our cryo-EM structure. AcrID1 prevents dsDNA R-loop formation by binding to the face of Cas10d, obstructing the path of the non-target strand towards the HD site. Subunits are colored as in Fig. 1.