Close

RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering

Jingwen Guan, Agnès Oromí-Bosch, Senén D. Mendoza, Shweta Karambelkar, Joel Berry, Joseph Bondy-Denomy

Preprint posted on 28 February 2022 https://www.biorxiv.org/content/10.1101/2022.02.14.480438v2

New CRISPR approach to make previously intractable phages engineerable

Selected by Mandy Neumann

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
Phage engineering approach
Figure 1: Scheme of phage engineering approach. The technique is divided into 3 steps: 1. The acrVIA1 gene flanked by homology arms integrates into the phage genome via homologous recombination in a bacterial cell harbouring the editing plasmid. 2. The phage population harvested from the first step contain both edited and non-edited phages. 3. The phage population is added to a bacterium that contains the Cas13a system with a crRNA targeting the insertion site. RNA transcripts of non-edited phages will by destroyed by the CRISPR system.

 

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

Posted on: 23 April 2022 , updated on: 25 April 2022

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

Read preprint (1 votes)

Author's response

Joseph Bondy-Denomy shared

Concerning the staining DNA with DAPI: how did you distinguish between phage DNA and bacterial DNA inside the bacterium?

phiKZ is known to destroy the bacterial genome within 10-15 min of infection and therefore it is quickly absent.

Do you have an idea whether the nucleus-like structure interferes or impacts the efficiency of homologous recombination?

Great question. We were worried about this and had planned strategies to drag plasmid DNA into the nucleus, but we quickly saw that HDR was working just fine. Clearly, the phage genome interacts with plasmid DNA during infection. Most phages do recombination well (especially those with UvsX recombinase) when exposed to plasmids or other phages, I suspect phiKZ is no different.

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 ?

Not in the short term. Our goal is to really enable our work on phiKZ, not to do any more tool development than we need for those goals. A recent preprint from Adler et al. in the Doudna lab really nicely showed how useful Cas13a can be for many other phages.

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?

In this paper, the anti-CRISPR is just an engineering trick, it isn’t there to block endogenous CRISPR systems in Pseudomonas. But in the case where one might engineer a phage with an Acr to protect it from native systems, of course it is reasonable to think that resistance could emerge. But I suspect receptor loss is a much easier thing for bacteria to do. It is actually pretty hard to evolve CRISPR-Cas mutations that escape an Acr mechanism, while maintaining CRISPR-Cas function.

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the genetics category:

Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University

This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.

 



List by Nándor Lipták

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf

 



List by Nándor Lipták

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)

 



List by Nándor Lipták

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar
Close