Extended Data Fig. 2: In-depth analysis of the dual-PAM prespacer bound GsuCas4/Cas1-Cas2 structure.
From: Mechanism for Cas4-assisted directional spacer acquisition in CRISPR–Cas
a. Comparison between the current 3.2 Å cryo-EM reconstruction with the previous negative staining reconstruction of the B. hal Cas4/1-2 complex (EMDB 20131)22. b–d. Pairwise alignment between GsuCas4/Cas1-Cas2/prespacer and EcoCas1-Cas2/prespacer8,31 (PDB 5DS4), EfaCas1-Cas2/prespacer12 (PDB 5XVN), and EfaCas1-Cas2/full-integration12 (PDB 5XVO), respectively. Alignments details are noted on the figure panel. Inset: the C-terminal tail of Cas2 plays similar roles in G. sul and E. fae structures in mediating edge-stacking with both Cas2 and Cas1. e. PAM was processed similarly in 22-bp or 26-bp mid-duplex containing prespacer by GsuCas4/Cas1-Cas2. f. SEC profile was similar when the two different prespacers were used to assemble the complex. g. Validation that prespacers containing a 22-bp mid-duplex are actively acquired in vivo. N=3 biologically independent assays were evaluated by PCR detection as shown, as well as relative percentages of expanded and non-expanded bands. Data presented as mean ± s.e.m.
