Characterization of the AcrIIC1 anti‒CRISPR protein for Cas9‒based genome engineering in E. coli.

Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr and CRISPR-Cas proteins.

Comparative genomic analysis of dwarf Vibrio myoviruses defines a conserved gene cluster for successful phage infection.

Tailed bacteriophages have been at the center of attention, not only for their ability to infect and kill pathogenic bacteria but also due to their peculiar and intriguing complex contractile tail structure. Tailed bacteriophages with contractile tails are known to have a Myoviridae morphotype and are members of the order Caudovirales. Large bacteriophages with a genome larger than 150 kbp have been studied for their ability to use multiple infection and lysis strategies to replicate more efficiently. On the other hand, smaller bacteriophages with fewer genes are represented in the GenBank database in greater numbers, and have several genes with unknown function. Isolation and molecular characterization of a newly reported bacteriophage named Athena1 revealed that it is a strongly lytic bacteriophage with a genome size of 39,826 bp. This prompted us to perform a comparative genomic analysis of Vibrio myoviruses with a genome size of no more than 50 kbp. The results revealed a pattern of genomic organization that includes sets of genes responsible for virion morphogenesis, replication/recombination of DNA, and lysis/lysogeny switching. By studying phylogenetic gene markers, we were able to draw conclusions about evolutionary events that shaped the genomic mosaicism of these phages, pinpointing the importance of a conserved organization of the genomic region encoding the baseplate protein for successful infection of Gram-negative bacteria. In addition, we propose the creation of new genera for dwarf Vibrio myoviruses. Comparative genomics of phages infecting aquatic bacteria could provide information that is useful for combating fish pathogens in aquaculture, using novel strategies.