Insights into Spacer Acquisition of the Type V-A CRISPR-Cas System of Francisella novicida U112

CRISPR-Cas systems immunise prokaryotic cells against foreign agents. In the process of CRISPR adaptation, short pieces of DNA from foreign elements are acquired and integrated into the CRISPR array. Here, we investigated the spacer acquisition mechanisms in the type V-A CRISPR-Cas system of Francisella tularensis subsp. novicida U112. We characterised the Cas1-Cas2 integrase in vitro and elucidated the sequence requirements of the pre-spacer and CRISPR array for optimal spacer incorporation. We also demonstrated that the active site of Cas2 coordinates metals and is important for full-site spacer integration. Furthermore, we established an in vivo spacer acquisition system in E. coli and found that all Cas proteins are required for efficient type V-A adaptation. We show that Cas12 greatly enhances adaptation efficiency in vivo. Spacers were preferentially acquired from plasmids encoding cas genes and genomic regions of prophages and origins of replication. We also found that Cas4 has 3'-5' exonuclease activity against ssDNA and ATP-independent unwinding activity towards dsDNA. Cas4 interacts with the Cas1-Cas2 complex and processes the 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 sequence, which is critical for invader targeting.