Type VI retrons need a phage to start making the DNA that switches on their toxin

Type VI retrons stood out because a signal was missing. "The way we typically detect retrons inside uninfected cells is by looking for this single-stranded DNA," said Karen Zhang, a Ph.D. student in Shipman's lab and lead author of the study ^[3]. Type VI retrons did not show it. "It was very odd, and we wondered if they might be broken," Zhang said ^[4].

The test that settled the question was functional. When the team tested certain bacteria carrying type VI retrons, the cells still defended themselves against phages ^[5]. A dead system would not have done that, so the missing signature pointed to a different design.

The experiments kept failing after that. "Whenever we tried the experiments we normally use to study retrons, they would fail, so we kept having to think of new ways to figure out how these retrons work," Shipman said ^[6]. The way through was Spacer-seq, a tool from Shipman's lab that uses components of the CRISPR-Cas immune system to keep a record of gene activity inside a cell ^[10]. "We had just about given up hope that any DNA was being made when this approach finally revealed a very, very low level of production in uninfected cells," Zhang said ^[11].

The team reports that type VI retrons do not make noticeable levels of DNA or toxin in uninfected cells ^[7]. That is a claim about levels, and Spacer-seq did find a very low one. What triggers the retron to start producing the DNA is a phage entering the cell ^[7].

"This is, by far, the strangest of the retron systems that we or anyone else has come across," Shipman said ^[13]. In older retrons the toxin is always present in uninfected cells and held in check by small single-stranded DNA molecules until a phage disturbs them ^[2]. In type VI, the DNA builds up and then interacts with an RNA that had been blocking toxin production, which lets toxin output ramp up ^[8]. "It's almost the mirror-image version of how we traditionally view retrons, because the DNA switches on toxin production instead of inhibiting an already produced toxin," Zhang said ^[12]. The toxin appears to be a tiny protein that embeds in the cell membrane and destabilizes the cell ^[9].

Retrons can efficiently modify and deliver single-stranded DNA, and Shipman's team is working to harness them to rewrite disease-causing mutations inside human cells ^[15]. Shipman said the type VI system uses "molecular mechanisms that we would never have thought to invent, but that we'll use in future technologies" ^[17]. The phys.org account quotes only Gladstone researchers and does not describe an editing experiment built from type VI parts. The tests it describes used bacteria carrying the retrons ^[5].