Characterization of Five CRISPR Systems in Microcystis aeruginosa FACHB-524 with Focus on the In Vitro Antiviral Activity of One CRISPR System

<i>Microcystis aeruginosa</i> is an important species causing cyanobacterial blooms, which can be effectively infected and lysed by cyanophages. Several strategies have been developed by <i>M. aeruginosa</i> to resist cyanophage infections, including the CRISPR-Cas systems. However, detailed information on the CRISPR-Cas systems in <i>M. aeruginosa</i> is rare. In the present study, the CRISPR-Cas systems of <i>M. aeruginosa</i> FACHB-524 were analyzed by genome re-sequencing, which showed that there are two type I (Cluster 1, I-B1; Cluster 2, I-D) and three type III-B (Cluster 3/4/5) CRISPR-Cas systems in the cyanobacteria. Further comparison revealed that spacer sequences of two type III-B systems targeted several genes of the cyanophage MaMV (<i>M. aeruginosa</i> myovirus) strains. One of the type III systems (Cluster 4) was then cloned and expressed in <i>Escherichia coli</i> BL21 (DE3). Protein purification and mass spectrometry identification revealed that a Cmr-crRNA effector complex formed in the <i>E. coli</i>. Subsequently, <i>T4 phage (T4)</i> was used to infect the <i>E. coli,</i> expressing the Cmr-crRNA complex with or without accessory proteins. The results showed that the Cmr-crRNA effector complex exhibited anti-phage activity and the accessory protein Csx1 enhanced the immune activity of the complex. Collectively, our results comprehensively demonstrate the CRISPR systems encoded by a strain of <i>M. aeruginosa</i>, and for the first time, one of the CRISPR systems was constructed into <i>E. coli</i>, providing a foundation for further in-depth analysis of cyanobacterial CRISPR systems.