Insights into spacer acquisition of the type V-A CRISPR–Cas system of <i>Francisella novicida</i> U112

CRISPR-Cas systems immunize prokaryotic cells through a CRISPR adaptation process, in which short DNA fragments from foreign elements are acquired and integrated into the CRISPR array. Here, we investigated the spacer acquisition mechanism of the type V-A CRISPR-Cas system from Francisella novicida U112. We characterized the Cas1-Cas2 integrase in vitro and elucidated the sequence requirements of the pre-spacer and the CRISPR array for optimal spacer incorporation. We demonstrated that Cas2 coordinates metals at its active site to facilitate full-site spacer integration. Furthermore, we introduced this spacer acquisition system into Escherichia coli cells and observed that, in vivo, all Cas proteins are required for efficient type V-A adaptation, with Cas12 significantly improving adaptation efficiency. We showed that spacers were acquired preferentially from plasmids encoding cas genes, and from genomic regions of prophages and origins of replication. In addition, we found that Cas4 possesses a 3'-5' exonuclease activity against single-stranded DNA and an ATP-independent unwinding activity towards double-stranded DNA. Cas4 interacts with the Cas1-Cas2 complex and processes pre-spacers in a PAM-dependent manner. The presence of Cas4 in vivo ensures that new spacers are derived from DNA immediately downstream of a 5'-TTTN-3' PAM, which is critical for targeting invaders.