Extended Data Fig. 7: In vitro assays to distinguish integration directionality.
From: Mechanism for Cas4-assisted directional spacer acquisition in CRISPR–Cas
a, b. Biochemistry showing that GsuCas4/1-2 is unable to integrate prespacer into the linear form of leader-repeat DNA. c. Successful prespacer integration into a leader-repeat containing plasmid by Cas4/1-2. d. The leader-repeat sequence cloned into the plasmid. We cleaved the leader-repeat sequence via the EcoRI and XhoI sites after the integration assay to further resolve the integration directionality on urea-PAGE. e. Schematic diagram explaining how the integration directionality can be resolved based on the fluorescent ssDNA sizes. f. Integration profile in urea-PAGE when both overhangs are integration-ready (7-nt long). Results showed that from the leader-repeat point of view, integration preferentially initiates from the leader-side, as the spacer-side integration trails after the leader-side integration in the time-course experiment. From the prespacer point of view, the integration directionality is scrambled. Each integration band contains two overlapping fluorescent signals. g. Native PAGE showing that in the concentration-gradient experiment, complex formation between Cas4/1-2 and prespacer takes place in a stepwise and PAM-dependent fashion.
