Mysterious DNA from Microbes Helps Defeat Viruses and Has Genome-Changing Potential | Science

By Elizabeth Pennisi11 November 2020, 16:45

Seven years ago, understanding nature inspired a revolutionary new technology, when researchers transformed a defense system used by bacteria to fight viruses into the gene-editing tool now known as CRISPR. But for another emerging genetic editor, understanding has delayed applications. For several years, researchers have adapted retrons – mysterious complexes of DNA, RNA and proteins found in certain bacteria – in a potentially powerful way to alter the genomes of single-celled organisms. Now, biology is catching up, as two groups report evidence that, like CRISPR, retrons 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, triggering self-destruction of newly infected cells so that the virus cannot replicate and spread to others. The Cell the paper “is the first to concretely determine a natural function for backrons,” says Anna Simon, a synthetic biologist at Strand Therapeutics who has studied bacterial oddities. Another document, which has so far appeared only as a prepress, reports a similar result.

The new understanding of the natural function of backs could increase efforts to put them to work. Retrons are “fairly efficient tools for accurate and efficient genome editing,” says Rotem Sorek, a microbial genomycist at the Weizmann Institute of Science and author of Cell she studies. But they still don’t rival CRISPR, in part because the technology wasn’t made to work in mammalian cells.

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

Similar constructs, dubbed retrons for reverse transcriptase, have been found in many bacteria. “They are truly an extraordinary biological entity, but no one knew what they were for,” says Ilya Finkelstein, a biophysicist at the University of Texas, Austin.

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

The team then noted that the DNA components encoding retron often accompanied a gene encoding the protein, and the protein ranged from retron to retron. The team decided to test their intuition that the sequence cluster represented a new phage defense. They went on to show that the bacteria needed all three components – reverse transcriptase, DNA-RNA hybrid, and the second protein – to defeat a variety of viruses.

How bacteria react

For a retron called Ec48, Sorek and colleagues showed that the associated protein delivers the final blow by reaching the outer membrane of a bacterium and altering its permeability. The researchers concluded that retron somehow “guards” another molecular complex that is the bacterium’s first antiviral line of defense. Some phages deactivate the complex, which triggers the retron to release the membrane-destroying protein and kill the infected cell, Millman, Sorek and their team reported Nov. 6 in Cell.

A second group came to similar conclusions. Led by Athanasios Typas, a microbiologist at the European Molecular Biology Laboratory (EMBL), Heidelberg, the group realized that alongside the genes that code for a retron in a Salmonella the bacterium was a gene for a toxic protein for Salmonella. The team found that retron normally keeps the toxin hidden 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 convergent our work was,” says Typas. The teams simultaneously published preprints of their work in June on bioRxiv. (The second group article is still under review in a journal.)

Even before these discoveries, other researchers had taken advantage of the then mysterious features of retrons to devise new gene editors. CRISPR easily targets and binds or cuts desired regions of the genome, but so far is not very adept at introducing new code into target DNA. Retrons, combined with elements of CRISPR, appear to be able to do better thanks to their reverse transcriptase: they can produce many copies of a desired sequence, which can be merged efficiently into the host’s genome. “Because CRISPR-based systems and retrons have different strengths, combining them is a very promising strategy,” says Simon.

In 2018, researchers at Hunter Fraser’s Stanford University lab introduced a basic retron-derived editor, dubbed CRISPEY (Cas9 retron precise parallel editing via homology). First, they created retrons whose RNA matched the yeast genes, but with a mutated base. They combined them with CRISPR’s “guide RNA,” found on the targeted DNA, and the CAS9 enzyme that acts as CRISPR’s molecular scissors. Once CAS9 cut the DNA, the cell’s DNA repair mechanisms replaced the yeast gene with DNA generated by retron reverse transcriptase.

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

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