Extended Data Fig. 8: In-depth analysis of the mechanistic coupling between half-integration and PAM cleavage by Cas4.

a. Time-course experiment showing ExoI trims PAM and non-PAM overhangs differently. The non-PAM 3′-overhang was trimmed to within one nucleotide of the preferred length, 7 nt. The PAM-side 3′-overhang was protected by the footprint of Cas4 in the same reaction. b. Time-course experiment resolving the order of events from prespacer processing to full integration. Using the Cas4/1-2 (left set) and Cas4/1-2 plus ExoI (middle set) lanes as controls, the right set of experiment shows ExoI trimming triggers the integration of the non-PAM overhang into the leader-proximal target DNA. This is followed by the stimulation of PAM cleavage, and then the full integration from PAM-overhang to spacer-side target. c. Temperature-dependency of PAM cleavage and spacer-side integration. d. Side-by-side comparison of PAM cleavage at 50 °C, prespacer alone or programmed to the half-integrated state. e. Quantification of the cleaved band in c. and d. revealing the elevated PAM cleavage and full integration when leader-side integration already took place. Data were collected from N = 3 biologically independent experiments and presented with mean ± s.e.m. Statistical significance was assessed by two-tailed t-test, with the exact P values displayed. f. Salt-dependency of PAM cleavage and full integration. g–i. Optimization of full integration reaction by defining its time course, Cas2-dependency, and pH-dependency, respectively. j. Defining pH-dependency of PAM cleavage by Cas4. k. SEC analysis of the Cas4/1-2 complex programmed with the half-integration product mimic. Samples in the integrated complex peak was used for cryo-EM data collection and single particle reconstruction. l, Schematics of the half-integration product mimic annealed from oligonucleotides. m. Urea-PAGE analysis of the SEC peak in k. revealing that Cas4/1-2 further catalyzed the full-integration reaction after binding to the half-integration mimic.