Spacer Acquisition Type V-A CRISPR-Cas system Francisella novicida

The ability to adapt to new threats is important for the virus-host interactions in microbial populations. CRISPR-Cas systems are able to immunize prokaryotic cells against foreign agents like bacteriophages and establish a sophisticated and adaptive defence towards specific predators. Crucial for this process, called adaptation, are the proteins Cas1 and Cas2, which integrate small spacer sequences of invading viruses into the hosts CRISPR array. While Cas1 and Cas2 are the core elements of the adaptation stage in almost all CRISPR-Cas systems, the occurrence of mechanistic variations during this process has been studied in only a few subtypes. Here, we describe how spacer acquisition functions in the type V-A CRISPR-Cas system of Francisella tularensis subsp. novicida U112 and show that this strain lacks a proper acquisition machinery due to a nonsense mutation in the cas4 gene. Cas4 proteins of different CRISPR-Cas types have recently been shown to facilitate PAM-dependent spacer selection and processing prior to integration. While a mutation in the active site of Cas4 in our system affected PAM-dependent spacer selection, it did not inhibit the integration of new spacers into the CRISPR locus by Cas1 and Cas2. We could characterize the type V-A Cas1-Cas2 integrase in vitro and elucidated the sequential requirements of the pre-spacer and the CRISPR array that are important for optimal spacer incorporation. Finally, we demonstrated the importance of Cas2 during integration and provided evidence that metal ion coordination by Cas2 is important for spacer integration in vivo and in vitro.