Engineering a phage's DNA by introducing modified CRISPR-Cas machinery genes

The science

Bacteriophages (or phages) are viruses that infect bacteria. They are the most abundant biological entity, and they can be found wherever bacteria live. To propagate themselves, phages inject their DNA into their bacterial hosts. Their DNA hijacks the host cell, forcing it to produce more copies of the phage until the cell bursts and releases thousands of new phages. In this study, researchers engineered a phage's DNA by introducing modified CRISPR-Cas machinery genes. This machinery uses a bacterial immune system as a genome editing tool. With this technology, the scientists precisely modified a bacterial host genome within a miniaturized laboratory microbial ecosystem called EcoFAB.

The impact

Most methods for editing bacterial genomes use circular molecules of DNA called plasmids to transfer DNA between bacterial cells. However, delivering plasmids to specific bacterial species within a mixed microbial community is often inefficient. On the other hand, phages are highly efficient at transferring DNA into specific bacterial species. This research leveraged these phages' properties to develop a new phage-based DNA delivery tool. The researchers also used this tool to precisely edit individual genes inside a target host organism within a living microbial community.

Summary

Scientists at North Carolina State University developed a single-step phage engineering method that takes advantage of the CRISPR-Cas9 system and a DNA recombination and repair mechanism. Using this method, the researchers engineered the genome of the phage lambda (λ) of Escherichia coli. They replaced a non-essential region of the λ genome with a selectable marker and an engineered base editor. This editor enabled precise modifications of the bacterial genomic DNA by converting one of the four DNA bases (or letters A, C, T, and G) into another. The researchers used this tool to convert a C into a T in specific genes, either within the bacterial chromosome or carried in a DNA plasmid. They demonstrated that the approach was successful by inactivating multiple genes, including the endogenous lacZ gene and plasmid-encoded fluorescent reporter and antibiotic resistance genes.

To demonstrate the effectiveness of this precise genome editing tool within a community context, the team used a fabricated ecosystem (EcoFAB) device. Recapitulating a soil microbial ecosystem, they filled the EcoFAB with sterile quartz sand and inoculated it with a soil bacterial community composed of E. coli and two other known bacterial species. They then added the engineered "editor" λ phage that can only infect E. coli to the EcoFAB. After incubating the phage with the bacterial community, they managed to base-edit 28 percent of the E. coli cells in the EcoFAB. These results highlight the potential of phages as DNA delivery vehicles for targeted members of a mixed soil community for precise genome editing.

Funding

Funding was provided by m-CAFEs Microbial Community Analysis & Functional Evaluation in Soils, a Science Focus Area led by Lawrence Berkeley National Laboratory and supported by the Department of Energy Office of Science, Office of Biological and Environmental Research.

Source:
Journal reference:

Nethery, M.A., et al. (2022) CRISPR-based engineering of phages for in situ bacterial base editing. PNAS. doi.org/10.1073/pnas.2206744119.

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