RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering

The viruses that infect bacteria, bacteriophages (or phages), possess numerous genes of unknown function. Genetic tools are required to understand their biology and enhance their efficacy as antimicrobials. Pseudomonas aeruginosa jumbo phage {Phi}KZ and its relatives are a broad host range phage family that assemble a proteinaceous "phage nucleus" structure during infection. Due to the phage nucleus, DNA-targeting CRISPR-Cas is ineffective against this phage and thus there are currently no reverse genetic tools for this family. Here, we develop a DNA phage genome editing technology using the RNA-targeting CRISPR-Cas13a enzyme as a selection tool, an anti-CRISPR gene (acrVIA1) as a selectable marker, and homologous recombination. Precise insertion of foreign genes, gene deletions, and the addition of chromosomal fluorescent tags into the {Phi}KZ genome were achieved. Deletion of phuZ, which encodes a tubulin-like protein that centers the phage nucleus during infection, led to the mispositioning of the phage nucleus but surprisingly had no impact on phage replication, despite a proposed role in capsid trafficking. A chromosomal fluorescent tag placed on gp93, a proposed "inner body" protein in the phage head revealed a protein that is injected with the phage genome, localizes with the maturing phage nucleus, and is massively synthesized around the phage nucleus late in infection. Successful editing of two other phages that resist DNA-targeting CRISPR-Cas systems [OMKO1 ({Phi}KZ-like) and PaMx41] demonstrates the flexibility of this method. RNA-targeting Cas13a system holds great promise for becoming a universal genetic editing tool for intractable phages. This phage genetic engineering platform enables the systematic study of phage genes of unknown function and the precise modification of phages for use in a variety of applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/480438v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@b1b4a6org.highwire.dtl.DTLVardef@cb0e08org.highwire.dtl.DTLVardef@1064217org.highwire.dtl.DTLVardef@1dcfbb5_HPS_FORMAT_FIGEXP M_FIG C_FIG