RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering

Background

For almost every organism on earth there is a virus that can infect it. Bacteriophages, viruses that attack bacteria, can be used to treat bacterial infections. Given the rise of antibiotic resistance among pathogenic bacteria, phage therapy is becoming increasingly popular. The bacterium Pseudomonas aeruginosa infects various organs, including the respiratory, urinary, and gastrointestinal tract. It is a multi-resistant “super-bug” for several reasons: it has an outer membrane with low permeability, which limits the uptake of antibiotics, it acquires antibiotic resistance genes through horizontal gene transfer, and it is highly adaptable to changing environmental conditions (Breidenstein et al. 2011). Patients with suppressed immune system and chronic lung diseases, such as cystic fibrosis, struggle with multi-resistant Pseudomonas infections, and phage therapy has the potential to cure these patients.

However, bacteria are not defenceless against bacteriophages: they have evolved multiple defence mechanisms, such as CRISPR-Cas systems, which are based on a library of CRISPR RNAs (crRNA) that recognize phage-specific genetic sequences. Upon invasion, the CRISPR system recognizes and cleaves phage’s genetic sequence. In the constant arms race between invader and defender, phages have acquired anti-CRISPR systems to block CRISPR systems.

In this preprint, the researchers use a combinatorial approach to engineer phages including the anti-CRISPR protein acrVIA1, which binds the CRISPR complex, thereby inhibiting nucleotide cleavage (Meeske et al. 2020) and promoting phage survival. Amongst others, the authors engineered the Pseudomonas-infecting giant phage φKZ.

Findings

Cas13a and acrVIA1 allow genetic engineering of φKZ

In a prior study, scientists discovered that the φKZ phage forms a proteinaceous structure around its genome, which blocks access for CRISPR systems. This protects the phage from DNA-targeting CRISPR systems and makes DNA-based genetic engineering of the phage impossible (Mendoza et al. 2020). Phage RNA transcripts travel out of the protein shield and are accessible to RNA-targeting CRISPR systems, such as Cas13a (Mendoza et al., 2020).

In this preprint, the authors exploit this loophole to engineer the genome of the φKZ phage. Their method allows editing and selecting for edited phages in three steps (figure 1):

1. Homologous recombination of the anti-CRISPR gene acrVIA1
2. Harvest phages
3. Select for edited phages using Cas13a

 

Insights in phage biology via genetic knockouts and reporter protein fusion

Equipped with this novel genome engineering approach, the authors set out to study phage biology by characterizing different gene functions. The scientists created gene deletions, translational fusions to fluorescent reporters, and genes insertions, including integration of barcodes.

The tubulin homologue PhuZ was previously hypothesized to be essential for phage assembly, shuttling phage capsid heads to the phage nucleus to load the genome into the capsid. However, this preprint refutes this hypothesis as the deletion of this gene yielded viable phages. It was observed that the phage nucleus lost its central position in the bacterium, in contrast to wildtype. The authors concluded that PhuZ anchors the phage nucleus in the middle of the bacterium but is not essential in laboratory conditions. Other gene knockouts turned out to be lethal, including orf54, whose product gp54 builds the phage nucleus, underlining the essentiality of these genes. Onto another protein that is very abundant in the phage’s capsid, they attached a fluorescent protein. This allowed them to track the protein’s journey over the course of a viral infection in a bacterium.

None of the engineered phages showed growth deficiencies or a reduced host range. This suggests that genome editing has no detrimental effect on the phage’s capability to infect its hosts.

Genome engineering of other phages

To verify that their editing technique is broadly applicable, the authors chose to engineer the jumbo phage OMKO1 and the podophage PaMx41. Both phages are resistant to DNA-targeting CRISPR systems and infect Pseudomonas. The jumbo phage OMKO1 is currently tested in a phase I/II clinical trial to treat antibiotic resistant Pseudomonas infections, hence a good example for phage therapy. Three quarters of the phage PaMx41 genes are unknown, which makes it a good example for basic research that focuses on phage biology.

Into OMKO1’s genome, the authors integrated 120 nucleotide-long barcodes together with acrVIA1. The inserted barcode did not impact the host range but did lead to a mild reduction in virulence. For the podophage PaMx41, they deleted a gene with unknown function. Taken together, they demonstrate that their method is broadly suitable for genetically engineering of previously intractable phages.

Importance and opinion

The researchers use an elegant system that combines three different biological processes to edit phages. The three elements target different molecules inside the cell: DNA for homologous recombination, RNA for the Cas13a nuclease and the Cas13a nuclease for the anti-CRISPR protein acrVIA1. This is a good example of how deep understanding of biological processes allows to develop new technologies. The presented three-step method allowed to engineer the genome of phages that were previously inaccessible to CRISPR-based genome engineering. By using an RNA-targeting CRISPR system, the authors could circumvent the nucleus-like barrier around the phage genome that previously prevented genome engineering. The chances are high that this system can engineer almost any phage. It is unlikely that phages involved Cas13a-targeting anti-CRISPR systems as Cas13a is rarely encoded in bacteria. Therefore, the authors expect that their method is broadly applicable to most phages. With an increasing number of phages that can be engineered, new possibilities in phage therapy will emerge. Engineering previously intractable phages will also be useful to gain new insights in phage biology, such as the mechanisms underlying phage-bacteria interaction as well as the function of uncharacterized phage genes.

Questions to the authors

• Concerning the staining DNA with DAPI: how did you distinguish between phage DNA and bacterial DNA inside the bacterium?
• Do you have an idea whether the nucleus-like structure interferes or impacts the efficiency of homologous recombination?
• In this study you focused on Pseudomonas-infecting phages, do you have plans to turn towards other phages that infect other multi-resistant bacteria such as enterohaemorrhagic Escherichia coli ?
• Supposing that phage therapy with anti-CRISPR systems will widely be applied to treat bacterial infections, resistance mechanisms against the anti-CRISPR will sooner or later arise and spread in bacteria. This will lead similarly to nowadays rising antibiotic resistances to untreatable infections. Do you have any ideas how to minimize, slow down and manage such developments?

References

Breidenstein, Elena B. M.; La Fuente-Núñez, César de; Hancock, Robert E. W. (2011): Pseudomonas aeruginosa: all roads lead to resistance. In: Trends in microbiology 19 (8), S. 419–426. DOI: 10.1016/j.tim.2011.04.005.

Meeske, Alexander J.; Jia, Ning; Cassel, Alice K.; Kozlova, Albina; Liao, Jingqiu; Wiedmann, Martin et al. (2020): A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity. In: Science (New York, N.Y.) 369 (6499), S. 54–59. DOI: 10.1126/science.abb6151.

Mendoza, Senén D.; Nieweglowska, Eliza S.; Govindarajan, Sutharsan; Leon, Lina M.; Berry, Joel D.; Tiwari, Anika et al. (2020): A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases. In: Nature 577 (7789), S. 244–248. DOI: 10.1038/s41586-019-1786-y.

Tags: anti-crispr, bacteriophages, crispr-cas, genome engineering, phage therapy

doi: https://doi.org/10.1242/prelights.31851

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