Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays

• Published in: Molecular cell Vol. 70; no. 1; pp. 48 - 59.e5
• Main Authors: Lee, Hayun, Zhou, Yi, Taylor, David W., Sashital, Dipali G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.04.2018
• Subjects: Cas1-Cas2; Cas4; CRISPR-Cas; DNA cleavage; DNA integration; negative stain EM; Cas4; CRISPR-Cas; Cas1-Cas2; DNA cleavage; DNA integration; negative stain EM
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2018.03.003

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3′ overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration. [Display omitted] •Cas4 forms a tight heterohexameric complex with the Cas1 spacer integrase•Cas4 is a Cas1-Cas2-dependent endonuclease that cleaves 3′ overhangs of prespacers•Cas4 cleavage is sequence and site specific and depends on the presence of a PAM•Cas4 blocks premature integration of uncleaved prespacers, ensuring spacer fidelity Lee et al. show that Cas4, a core family of CRISPR-associated proteins, associates with the Cas1 spacer integrase and is required for efficient prespacer processing during CRISPR-Cas adaptation. Cas4 processing prevents non-functional spacers from being integrated into the CRISPR array, ensuring the fidelity of the adaptation process.