Seven years ago, an understanding of nature inspired a revolutionary new technology, when researchers turned a defense system used by bacteria to thwart viruses into the gene-editing tool now known as CRISPR. But for another emerging gene editor the understanding has lagged the applications. For several years, researchers have been adapting retrons—mysterious complexes of DNA, RNA, and protein found in some bacteria—into a potentially powerful way to alter genomes of single cell organisms. Now, biology is catching up, as two groups report evidence that, like CRISPR, retrons are part of the bacterial immune arsenal, protecting the microbes from viruses called phages.
Last week in Cell, one team described how a specific retron defends bacteria, triggering newly infected cells to self-destruct so the virus can’t replicate and spread to others. The Cell paper “is the first to concretely determine a natural function for retrons,” says Anna Simon, a synthetic biologist at Strand Therapeutics who has studied the bacterial oddities. Another paper, which so far has appeared only as a preprint, reports a similar finding.
The new understanding of retrons’ natural function could boost efforts to put them to work. Retrons are “quite efficient tools for accurate and efficient genome editing,” says Rotem Sorek, a microbial genomicist at the Weizmann Institute of Science and an author of the Cell study. But they don’t rival CRISPR yet, in part because the technology hasn’t been made to work in mammalian cells.
In the 1980s, researchers studying a soil bacterium were puzzled to find many copies of short sequences of single-stranded DNA littering the cells. The mystery deepened when they learned each bit of DNA was attached to an RNA with a complementary base sequence. Eventually they realized 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 the reverse transcriptase, were found in many bacteria. “They really are a remarkable biological entity, yet nobody knew what they were for,” says Ilya Finkelstein, a biophysicist at the University of Texas, Austin.
Sorek came upon an early hint of their function when he and his colleagues searched through 38,000 bacterial genomes for genes used to fight off phages. Such genes tend to be close to one another, and his team developed a computer program that searched for new defense systems next to the genes for the CRISPR and other known antiviral constructs. One stretch of DNA stood out to Weizmann graduate student Adi Millman because it included a gene for a reverse transcriptase flanked by stretches of DNA that didn’t code for any known bacterial proteins. By chance, she came across a paper about retrons and realized that the mysterious sequences encoded one of their RNA components. “That was a nontrivial leap,” Sorek says.
The team then noticed that the DNA encoding retron components often accompanied a protein-coding gene, and the protein varied from retron to retron. The team decided to test its hunch that the cluster of sequences represented a new phage defense. They went on to show that bacteria needed all three components—reverse transcriptase, the DNA-RNA hybrid, and the second protein—to defeat a variety of viruses.
For a retron called Ec48, Sorek and colleagues showed the associated protein delivers the coup de grâce by homing in on a bacterium’s outer membrane and altering its permeability. The researchers concluded that the retron somehow “guards” another molecular complex that is the bacterium’s first line of antiviral defense. Some phages deactivate the complex, which triggers the retron to unleash the membrane-destroying protein and kill the infected cell, Millman, Sorek, and their team reported on 6 November in Cell.
A second group has reached similar conclusions. Led by Athanasios Typas, a microbiologist at the European Molecular Biology Laboratory (EMBL), Heidelberg, the group realized that next to the genes coding for a retron in a Salmonella bacterium was a gene for a protein toxic to Salmonella. The team discovered the retron normally keeps the toxin under wraps, 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,” Typas says. The teams simultaneously posted preprints on their work in June on bioRxiv. (The second group’s paper is still under review at a journal.)
Even before these discoveries, other researchers had taken advantage of retrons’ then-mysterious features to devise new gene editors. CRISPR easily targets and binds to or cuts desired regions of the genome, but so far it isn’t very adept at introducing new code in the target DNA. Retrons, combined with elements of CRISPR, seem able to do better thanks to their reverse transcriptases: They can manufacture lots of copies of a desired sequence, which can be spliced efficiently into the host genome. “Because CRISPR-based systems and retrons have different strengths, combining them is a highly promising strategy,” Simon says.
In 2018, researchers in Hunter Fraser’s Stanford University lab introduced a retron-derived base editor, dubbed CRISPEY (Cas9 retron precise parallel editing via homology). First, they made retrons whose RNA matched yeast genes, but with one base mutated. They combined them with CRISPR’s “guide RNA,” which homes 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 the DNA generated by the retron’s reverse transcriptase.
CRISPEY enabled Stanford graduate student Shi-An Anderson Chen and his colleagues to efficiently make tens of thousands of yeast mutants, each different by just one base. That let them determine, for example, which bases were essential for yeast to thrive in glucose. “CRISPEY is very cool and extremely powerful,” says Harmit Malik, an evolutionary biologist at the Fred Hutchinson Cancer Research Center. This year, two other teams—led by geneticist George Church at Harvard University and Massachusetts Institute of Technology synthetic biologist Timothy Lu—described similar feats in bacteria in bioRxiv preprints.
Researchers are excited about retrons, but caution they have a lot to learn about how to turn these bacterial swords into plowshares. “It could be that retrons will be as revolutionary as CRISPR has been,” Simon says. “But until we understand more about the natural biology and synthetic behavior of retrons, it is difficult to say.”