Mechanism for Cas4-assisted directional spacer acquisition in CRISPR–Cas

• Published in: Nature (London) 2021-10, Vol.598 (7881), p.515-520
• Main Authors: Hu, Chunyi, Almendros, Cristóbal, Nam, Ki Hyun, Costa, Ana Rita, Vink, Jochem N. A., Haagsma, Anna C., Bagde, Saket R., Brouns, Stan J. J., Ke, Ailong
• Format: Article
• Language: English
• Subjects: 101/28; 45; 631/326/1320; 631/337/1644; 631/45/535/1258/1259; 631/45/607/1167; Biosynthesis; Constipation; CRISPR; CRISPR-Associated Proteins - metabolism; CRISPR-Cas Systems; Databases, Genetic; Deoxyribonucleic acid; Dissociation; DNA; Endonucleases - metabolism; Genetic research; Geobacter - enzymology; Humanities and Social Sciences; Insertion; Integrase; Integration; Interfaces; Microscopy; Models, Molecular; Molecular Conformation; Molecular interactions; multidisciplinary; Nuclease; Nucleotide Motifs; Nucleotide sequence; Prokaryotes; Restriction enzymes, DNA; Science; Science (multidisciplinary); Spacer; Spacers
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/s41586-021-03951-z

Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array 1 . Spacer insertion is carried out by the Cas1–Cas2 integrase complex 2 – 4 . A substantial fraction of CRISPR–Cas systems use a Fe–S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM) 5 , 6 and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens , in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3′-overhang trigger Cas4/Cas1–Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This ‘molecular constipation’ prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1–Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality. Structures of the Cas4–Cas1–Cas2 complex from Geobacter sulfurreducens show that a 3′-overhang in the protospacer adjacent motif is required for complex assembly and spacer insertion into the CRISPR array.