Retrons raise the bar for gene research

Within bacterial cells, specialized immune systems known as retrons fend off viral attacks. But that’s not all they can do. Scientists, including Seth Shipman, Ph.D., at Gladstone Institutes, have shown that retrons also serve an important purpose in the lab: precise DNA editing. In fact, retrons can be combined with CRISPR—the far more famous bacterial-defense-system-turned-gene-editor—to better edit human cells.

Yet, for all their potential to help scientists better understand disease and develop new therapies, only a small number of retrons have been studied. In nature, many thousands of variations exist, some with greater gene-editing powers than others.

In a study published in Nature Biotechnology, Shipman and his team greatly expand the universe of retron knowledge. They carried out a “census” of 163 never-before-tested retrons and identified many that can edit DNA more quickly and efficiently than those currently used in research.

“The baseline for retron technology has now significantly improved,” says Shipman, lead author of the new study. “In nearly every cell type, we found retrons that were much better editors than what we had started with.”

The scientists tested the new retrons in bacteria, phages (viruses that infect bacteria), and human cells. Some of the newly characterized retrons, they discovered, can edit efficiently enough to be used to treat disease. Their findings also provide an extensive database to start understanding the different properties of different retrons.

Expanding a toolkit

Ever since the advent of CRISPR more than a decade ago, scientists have used gene editing to more fully understand how cells function and to develop new ways to treat—or even cure—diseases. Retrons, with their natural ability to produce large amounts of DNA to detect invading viruses, take this capability to a new level.

Shipman is among the global leaders in the development of retrons as a biotechnology tool. He uses them as tiny factories inside cells to create the strands of DNA needed for gene editing.

Even though retrons are very common in bacteria, scientists have only been using a handful of retrons first described in the 1980s. A few years ago, researchers in Spain looked through genome databases and identified nearly 2,000 stretches of DNA they predicted to be retrons.

“Nobody had tested these retrons in a lab, so we decided to do just that,” says Asim Khan, co-first author of the new study and a former research associate in Shipman’s lab who is now pursuing his Ph.D. at Columbia University.

“We started taking retrons from that list and putting them into cells to see which ones would work and which ones would be useful for technology, and whether there are interesting differences between them.”

In the new work, Shipman’s group carried out lab experiments on a sampling of 163 of the presumed retrons. The retrons were selected to capture the diversity of the Spanish team’s complete list.

The researchers engineered E. coli bacteria to contain the code for each predicted retron, and tested whether the sequences were truly retrons that could produce DNA. They confirmed that most of them were indeed functional retrons.

“We then wanted to determine whether any of the new retrons could be better editors than the standard ones we’ve been using, so we measured how much DNA each new retron could produce,” says co-first author of the work Matías Rojas-Montero, a former research associate in the Shipman lab who is pursuing his Ph.D. at UC San Francisco (UCSF).

“Our lab previously found that the amount of DNA produced by a retron is a good predictor of how well it will work in genome editing.”

The team discovered that relationships between retrons predicted how much DNA they produced—those that produced the most tended to be related retron systems. Moving forward, this knowledge could help scientists predict how effective other retrons would be for editing.

Better editors

Once Shipman’s group verified that the retrons could produce DNA in E. coli, they selected 29 retrons to test for their ability to work with gene editing systems in E. coli and phages.

In E. coli, eight of the retrons led to better editing rates than Retron-Eco1, which is currently considered the gold-standard retron for gene editing in bacteria. One retron, from the bacteria Klebsiella variicola, had a 10-fold increase in editing efficiency compared to Eco1. In phages, four retrons were more efficient than Eco1.

Finally, the team tested more than 130 retrons for their ability to carry out gene editing in human cells; 58 of those had higher rates of precise editing than Eco1. The top-performing retrons, they showed, made correct edits in 30 to 40 percent of cells. This editing rate is considered high enough to make a therapeutic difference for many diseases that researchers would like to treat with gene therapy, such as autoimmune diseases and blood disorders.

Shipman and his colleagues are planning additional studies on the new retrons—as well as predicted retrons that didn’t yield DNA—to better understand what parts of the retrons influence their DNA production and ability to work with gene editors. Ultimately, they hope the list provides researchers with new, more effective options when they set out to edit genes using retrons.

“In addition to using the retrons ourselves to edit genomes and introduce genetic variants that are related to diseases, we’ve already been giving these new retrons out to many other labs that are excited about using them,” says Shipman, also an associate professor in the Department of Bioengineering and Therapeutic Sciences at UCSF, as well as a Chan Zuckerberg Biohub Investigator.

“As we keep digging deep into the properties of these retrons, we think we’ll be able to engineer and improve them even more.”