CRISPR-Cas immunity protects prokaryotes against foreign genetic elements. CRISPR-Cas uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers). It remains elusive how Cas1-Cas2 acquires spacers from cellular DNA fragments (prespacers) and how it integrates them into the CRISPR array in the correct orientation. By using the high spatiotemporal resolution of single-molecule fluorescence, we reveal that Cas1-Cas2 obtains prespacers in various forms including

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... nded DNA and partial duplexes by selecting them in the DNA-length and PAM-dependent manner. Furthermore, we identify DnaQ exonucleases as enzymes that can mature the Cas1- Cas2-loaded precursor prespacers into an integration-competent size. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and subsequent spacer integration in the correct orientation. This kinetic coordination in prespacer selection and PAM trimming provides comprehensive understanding of the mechanisms that underlie the integration of functional spacers in the CRISPR array.