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. Author manuscript; available in PMC: 2022 Jun 4.
Published in final edited form as: Nature. 2021 Sep 29;598(7881):515–520. doi: 10.1038/s41586-021-03951-z

Figure 3. Mechanistic insights from the single-PAM prespacer bound GsuCas4/Cas1-Cas2 structure.

Figure 3.

a. Cryo-EM density and structure of the single-PAM prespacer bound GsuCas4/1-2 complex. Cas4 is absent from the non-PAM side. b. ExoI is capable of trimming the non-PAM overhang to the optimal length for integration; PAM-side is protected by Cas4. c. In vitro integration assay setup and the expected readout. d. Non-PAM overhang is unidirectionally integrated to the leader-proximal end of the CRISPR repeat after ExoI trimming. e. Cryo-EM density and structure of a sub-complex. Cas4/1 dimer is missing from the non-PAM side. f. EMSA showing Cas4/1-2 is assembled sequentially, and preferentially on PAM-containing prespacers. g. Mechanistic model explaining Cas4-dependent prespacer biogenesis and directional integration.