CRISPR kick-started a golden age of genetic research — but in nature, there are hundreds of similar systems with unexplored potential for gene editing. Now, scientists have made huge strides in explaining how an enigmatic system called SPARDA works.
CRISPR systems have enabled scientists to edit genetic information more easily than ever before. Although it’s best known for its use in gene editing, CRISPR is actually an adapted bacterial immune defense system that was repurposed for human use.
Molecular argonautes
Study co-author Mindaugas Zaremba, a biochemist at Vilnius University in Lithuania, told Live Science that before the new work, researchers had conducted only limited studies of SPARDA systems. They had established that the proteins that make up the system adopt a kamikaze-like approach to cell defense, guarding the wider population of bacteria against foreign DNA, including free-floating DNA called plasmids and viruses called phages.
“SPARDA systems were demonstrated to protect bacteria from plasmids and phages by degrading the DNA of both infected cells and invaders, thereby killing the host cell but at the same time preventing further spread of the infection within the bacterial population,” Zaremba said.
How SPARDA worked at a molecular level remained unclear, prompting Zaremba and his team to use the AI protein analysis tool AlphaFold, among a suite of other analysis techniques, to dig into SPARDA’s setup. AlphaFold uses machine learning to predict the 3D shape of proteins based on the sequence of their underlying building blocks.
The SPARDA system is built from argonaute proteins, named for their resemblance to argonaut octopuses (Argonauta). The proteins were originally identified in plants, where seedlings affected by mutations in these proteins developed narrow leaves that reminded scientists of an octopus’s tentacles. These argonaute proteins are evolutionarily conserved and are present in cells across the three kingdoms of life.
Zaremba’s analysis looked at SPARDA systems randomly selected from two different bacteria. The first, Xanthobacter autotrophicus, is a soil-dwelling microbe that shuns sunlight and builds its food from locally sourced nitrogen. The second, Enhydrobacter aerosaccus, was first found in Michigan’s Wintergreen Lake and has built-in airbags that help it float around watery environments.
Zaremba’s team chopped the SPARDA systems out of these bacteria and placed them in the reliable model organism E. coli for study. A molecular analysis revealed that each of their argonaute proteins included a critical “activating region.” They called this area the beta-relay, because it resembled electrical relay switches that control machinery by flicking between “on” or “off” states.
When the SPARDA systems detected external threats, these switches changed shape. The new shape enabled the proteins to form complexes with other activated argonaute proteins. When that happens, the proteins line up like soldiers on parade, forming long, spiraling chains. These chains chop up any surrounding DNA that they encounter in an extreme reaction that spares neither the host nor the invader. This stops the infection from spreading to other cells.
Zaremba’s team then used AlphaFold to scan for beta-relays in similar bacterial proteins. The same switches popped up repeatedly, suggesting that the relays are a universal feature of this protein type.
SPARDA in diagnostics
SPARDA is essential for bacterial defense, but Zaremba’s team argues that the system could also help humans.
Activating SPARDA is a last-ditch maneuver for bacterial cells, to be used only when an infection is definitively present. Therefore, the system includes an incredibly accurate recognition system for spotting foreign DNA that would warrant self-destruction.
Researchers could repurpose the system for diagnostics, Zaremba suggested. In that scenario, the beta-relay could be altered to be activated only when a genetic sequence of interest is identified — so it would react only to the genetic material of a flu virus or SARS-CoV-2, for instance. This mechanism underlies existing CRISPR-based diagnostic tools.
The CRISPR diagnostics, however, are currently limited in their function — they recognize targets only when certain DNA sequences, called PAM sequences, flank them. These sequences are like the prongs on the end of a plug; if they don’t match a socket, the system will have no power. This means choosing the right CRISPR protein to match a particular target is essential.
“We already know that SPARDA systems do not require a PAM sequence,” Zaremba said. This means they could act like a universal adapter, giving future DNA diagnostics more flexibility and ultimately making the tests better at detecting a range of germs.
CRISPR research won a Nobel Prize and changed science forever. While SPARDA research is at a far earlier stage of research, its inner workings suggest that the design of tiny organisms could hold lessons for the biggest questions in science.