Genetic “hitchhikers” can be targeted with CRISPR

In a new study, North Carolina State University researchers characterize a set of molecular tools for rewriting, not just editing, large stretches of an organism’s DNA based on CRISPR-Cas systems linked to selfish genetic “autopilots” called transposons.

Researchers are exploring different IF CRISPR-Cas systems and engineering them to add genetic cargo—up to 10,000 extra letters of genetic code—to the transposon cargo to make the desired changes in a bacterium—in this case, E. coli. The paper appears in Nucleic acid research.

The findings expand the CRISPR toolbox and could have significant implications for manipulating bacteria and other organisms at a time when flexible genome editing is needed in therapeutics, biotechnology, and more sustainable and efficient agriculture.

Bacteria use CRISPR-Cas as an adaptive immune system to resist attacks from enemies such as viruses. These systems have been adapted by scientists to delete or cut out and replace certain sequences of the genetic code in different organisms. The new finding shows that exponentially more genetic code can be moved or added, potentially increasing the functionality of CRISPR.

“In nature, transposons have co-opted CRISPR systems to selfishly move around an organism’s genome to help themselves survive. We in turn co-opt what happens in nature by integrating with transposons a programmable CRISPR-Cas system that can move genetic cargo that we engineer to perform a specific function,” said Rodolphe Barango, emeritus professor of food, bioprocessing and of Nutrition at North Carolina State and corresponding author of the paper describing the study.

“Using this method, we have shown that we can create genomes by moving pieces of DNA up to 10,000 letters long,” Barangu said. “Nature already does this – bioinformatics data shows examples of up to 100,000 genetic letters being moved by transposon-based CRISPR systems – but now we can control and engineer it with this system.

“To complete the hitchhiking analogy, we design the hitchhiker to deliver a particular piece of luggage or cargo to the car to deliver some type of payload when the car arrives at its destination.”

The study shows that the researchers are proving the effectiveness of the method both in vitro on the lab bench and in vivo on E. coli. The researchers selected 10 different transposons associated with CRISPR to test the effectiveness of the method. This approach worked with all 10 transposons, although their effectiveness varied depending on factors such as temperature and size transposon‘s cargo load.

“It was interesting to find that all the systems we tested were functional after being reconstructed into genome-editing tools from their native biological forms,” ​​said Avery Roberts, a graduate student at NC State and first author of the study. “We have discovered new possibilities for these systems, but there are likely to be many more related discoveries and applications as the field moves at a rapid pace.”

The study also showed that the method can be used with different transposons simultaneously.

“Instead of a single gene—as is the case with other CRISPR systems, such as the more common type II Cas-9 system—we can introduce an entire metabolic pathway to enable a whole new set of functions in the body,” Barangu said. “In the future, this could mean, for example, giving plants more flexible resistance to disease or drought.”

“We are excited by these findings and see the potential to apply these newly discovered systems in agricultural plants to accelerate the development of more resistant and high-yielding varieties,” said Gusui Wu, global head of seed research at Syngenta Seeds.

Barangou and Wu add that the work in this research provides an excellent example of a public-private partnership that drives scientific discovery and trains tomorrow’s workforce.

Co-authors on the paper include NC State graduate student Avery Roberts and former NC State Ph.D. student Matthew Nethery.