Extended Data Fig. 1: Reconstitution and characterization of the GsuCas4/Cas1-Cas2 complex.
From: Mechanism for Cas4-assisted directional spacer acquisition in CRISPR–Cas
a. Active site substitution in Cas4 nuclease center (H48G, D100A) reduced in vivo spacer acquisition efficiency dramatically. Left three panels display the WebLogo of PAM code from spacers integrated by each Cas4/1-2 variant. Rightmost panel displays the number of deep-sequencing reads that confirm spacer integration. b–d. GsuCas4/1 purification analyzed by SDS-PAGE, coloring from the Fe-S cluster, and SEC profile, respectively. e,f. Affinity purification of GsuCas2, SDS-PAGE, and SEC analysis, respectively. g. GST pull-down experiments revealing the physical interaction between GsuCas4/1 and GsuCas2, with or without prespacer present. h. Metal ion dependency in PAM cleavage reaction. i. Biochemistry showing Cas4/1-2 specifically cleaves the PAM-embedded 3′-overhang in prespacer. j. PAM-cleavage specificity is lost over time, presumably due to Fe-S oxidation in Cas4. k. SEC profile of GsuCas4/Cas1-Cas2, alone or programmed with different prespacer substrates. PAM-containing prespacers drive high-order complex formation. l. Cryo-electron micrographs of three different complexes, with corresponding preliminary 2D averages to investigate sample quality.
