Mysterious Microbial DNA Helps Defeat Viruses, Has Genome Editing Potential | Science

By Elizabeth PennisiNov 11, 2020, 4:45 PM

Seven years ago, an understanding of nature inspired a revolutionary new technology, when researchers transformed a defense system used by bacteria to thwart viruses into a gene-editing tool now known as CRISPR. . But for another emerging gene editor, understanding has lagged behind in applications. For several years, researchers have been adapting retons – mysterious complexes of DNA, RNA, and proteins found in certain bacteria – into a potentially powerful way to modify the genomes of single-celled organisms. Now, biology is catching up, as two groups report evidence that, like CRISPR, retons are part of the bacterial immune arsenal, protecting microbes from viruses called phages.

Last week in Cell, a team described how a specific retron defends bacteria, causing newly infected cells to self-destruct so the virus cannot replicate and spread to others. the Cell The article “is the first to actually determine a natural function of retons,” says Anna Simon, a synthetic biologist at Strand Therapeutics who has studied bacterial oddities. Another article, which so far has only appeared in pre-print form, reports a similar finding.

New understanding of the natural function of retons could stimulate efforts to implement them. Retrons are “quite effective tools for precise and efficient genome editing,” says Rotem Sorek, a microbial genomicist at the Weizmann Institute of Science and author of the Cell study. But they don’t yet compete with CRISPR, in part because the technology wasn’t designed to work in mammalian cells.

In the 1980s, researchers studying soil bacteria were intrigued to find many copies of short, single-stranded DNA sequences littering cells. The mystery deepened when they learned that every bit of DNA was attached to an RNA with a complementary base sequence. Eventually, they realized that an enzyme called reverse transcriptase had made this DNA from the attached RNA and that the three molecules – RNA, DNA and enzyme – formed a complex.

Similar constructs, called retons for reverse transcriptase, have been found in many bacteria. “They really are a remarkable biological entity, but no one knew what they were used for,” says Ilya Finkelstein, a biophysicist at the University of Texas at Austin.

Sorek came across a first indication of their function when he and his colleagues searched 38,000 bacterial genomes for genes used to fight phages. These genes tend to be close to each other, and his team developed a computer program that searched for new defense systems alongside genes for CRISPR and other known antiviral constructs. One DNA segment stood out for Weizmann graduate student Adi Millman because it included a gene for reverse transcriptase flanked by DNA segments that did not encode any known bacterial proteins. By chance, she stumbled upon an article on retons and realized that the mysterious sequences encode one of their RNA components. “It was a non-trivial jump,” Sorek says.

The team then noticed that the DNA encoding the components of the retron often accompanied a gene encoding the protein and that the protein varied from one retron to another. The team decided to test their intuition that the group of sequences represented a new defense against phages. They then showed that bacteria need all three components – reverse transcriptase, the DNA-RNA hybrid, and the second protein – to defeat a variety of viruses.

How bacteria fight back

For a retron called Ec48, Sorek and his colleagues have shown that the associated protein provides the final blow by attaching itself to the outer membrane of a bacteria and modifying its permeability. The researchers concluded that the retron somehow “protects” another molecular complex that is the bacteria’s first line of antiviral defense. Some phages deactivate the complex, which triggers the retron to release the membrane killing protein and kill the infected cell, Millman, Sorek and their team reported on November 6 at Cell.

A second group came to similar conclusions. Led by Athanasios Typas, a microbiologist at the European Laboratory for Molecular Biology (EMBL), Heidelberg, the group realized that alongside genes encoding a retron in a Salmonella bacteria was a gene for a protein toxic to Salmonella. The team found that the retron normally keeps the toxin enveloped, but activates it in the presence of phage proteins.

The two groups met at an EMBL meeting in the summer of 2019. “It was refreshing to see how complementary and converging our work was,” says Typas. The teams simultaneously posted pre-impressions of their work in June on bioRxiv. (The article in Group 2 is still under review in a journal.)

Even before these discoveries, other researchers had taken advantage of the then mysterious characteristics of retons to design new gene editors. CRISPR targets and readily binds or cuts to desired regions of the genome, but so far it has not been very good at introducing new code into target DNA. Retrons, combined with elements of CRISPR, appear to be able to do better thanks to their reverse transcriptases: they can make many copies of a desired sequence, which can be efficiently spliced ​​into the host genome. “Because CRISPR-based systems and retrons have different strengths, combining them is a very promising strategy,” says Simon.

In 2018, researchers at Stanford University’s Hunter Fraser lab introduced a retron-derived base editor dubbed CRISPEY (Accurate Parallel Editing of the Cas9 Retron via Homology). First, they made retons whose RNA matched yeast genes, but with a mutated base. They combined them with CRISPR’s “guide RNA”, which harbors the targeted DNA, and the CAS9 enzyme, which acts like the molecular scissors of CRISPR. Once CAS9 cut the DNA, the cell’s DNA repair mechanisms replaced the yeast gene with DNA generated by the reverse transcriptase of the retron.

CRISPEY has enabled Stanford graduate student Shi-An Anderson Chen and his colleagues to efficiently manufacture tens of thousands of yeast mutants, each different from a single base. This allowed them to determine, for example, which bases were essential for yeast to thrive in glucose. “CRISPEY is very cool and extremely powerful,” says Harmit Malik, evolutionary biologist at the Fred Hutchinson Cancer Research Center. This year, two more teams – led by Harvard University geneticist George Church and Massachusetts Institute of Technology synthetic biologist Timothy Lu – described similar exploits in bacteria in bioRxiv pre-prints.

Researchers are excited about retons, but beware, they have a lot to learn about how to turn these bacterial swords into plowshares. “It could be that retons are as revolutionary as CRISPR was,” says Simon. “But until we understand more about the natural biology and synthetic behavior of retons, it’s hard to say.