RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering

Abstract

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 ΦKZ 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 ΦKZ 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 (ΦKZ-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.

Bacteriophages antagonize cGAS-like immunity in bacteria

“…By contrast, the PaMx41 escaper phage and PaMx33, PaMx35, and PaMx43 phages exhibited high titer on all strains (Figure 2C, S2), demonstrating that the short protein is not a dominant CBASS activator. We next deleted orf24 from PaMx41, a PaMx41- orf24 X37Q escaper, PaMx33, PaMx35, and PaMx43 using Cas13a genome engineering technology (Guan et al, 2022) (Figure 3A). The titer of the Δorf24 phages was reduced 2-5 orders of magnitude on Pa011 WT bacterial lawns relative to their plaquing on the ΔCBASS lawn, which could be complemented with the long gp24 expressed in trans (Figure 3B).…”

“…Construction of template plasmids for homologous recombination and selection of engineered phages via the CRISPR-Cas13a system were performed as described previously (Guan et al, 2022). Specifically, homology arms of >500bp up- and downstream of PaMx41 acbIIA1 were amplified by PCR using PaMx41 WT phage genomic DNA as the template.…”

RNA-targeting CRISPR-Cas13 Provides Broad-spectrum Phage Immunity

“…Notably, neither Cas13 variant has been investigated for antiviral activity. While a Cas13a ortholog from Listeria seeligeri has been used to restrict temperate and nucleus-forming phages 19, 30, 32, 34 , LbuCas13a comes from a phylogenetically distinct sub-clade of Cas13a effectors (Supplementary Fig. 1).…”

“…Leveraging the broad vulnerability of phages to Cas13a-targeting, we demonstrated how this robust counterselection could be employed to enrich markerless genome edits in E. coli phage. Most Cas-based counterselection methods show extensive crRNA or phage variability 25,28,34,47 , rely on native CRISPR host-biology [46][47][48][49] , and/or yield a high rate of escape mutants 34,46 . In contrast, we observed little variability in Cas13a counterselection efficacy across the eight phages and 21 crRNAs tested in this study.…”

“…Upon target RNA binding, Cas13 unleashes general, non-specific RNA degradation that arrests growth of the virocell to block infection progression, thereby limiting infection of neighboring cells 30 . Since all known viruses produce RNA 31 , Cas13 is capable of inhibiting dsDNA phages, primarily shown through studies investigating temperate 30, 32, 33 and nucleus forming 19, 34 phages. However, other Class 2 CRISPR effectors have encountered serious limitations in overcoming the diversity of genetic content encoded in phages 12, 19, 20, 35 .…”