Gene-editing breakthrough: CRISPR hitchhikers can rewrite, not just change, code

RALEIGH – In a new study, researchers at North Carolina State University characterize a set of molecular tools for rewriting — not just modifying — large chunks of an organism’s DNA, based on CRISPR-Cas systems paired with selfish genetic “hitchhikers” called transposons.

Researchers study various IF-type CRISPR-Cas systems and engineer them to add genetic cargo — up to 10,000 additional genetic code letters — to the transposon cargo to make desired changes to a bacterium — in this case, Escherichia coli.

The findings expand the CRISPR toolbox and could have significant implications for the manipulation of 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 remove or cut and replace specific sequences of genetic code in a variety of organisms. The new finding shows that exponentially larger amounts of genetic code can be moved or added, potentially increasing the functionality of CRISPR.

Rodolphe Barrangou (NCSU photo)

“In nature, transposons have co-opted CRISPR systems to selfishly move around an organism’s genome to help themselves survive. In turn we are co-opting what happens in nature by integrating with transposons a programmable CRISPR-Cas system that can move around the genetic cargo we design to perform a certain function,” said Rodolphe Barrangou, Todd R. Klaenhammer Distinguished Professor of Food, Bioprocessing and Nutrition Sciences at NC State and corresponding author of a paper describing the research.

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

Gene editing advances: NC State researchers use CRISPR to turn the tables on bacteria

“To complete the hitchhiking analogy, we are designing the hitchhiker to carry certain baggage or cargo into the car to deliver a certain type of payload when the car arrives at its destination.”

The study shows the researchers demonstrating the effectiveness of the method both in vitro on the laboratory bench and in vivo Escherichia coli. The researchers selected 10 different CRISPR-associated transposons to test the effectiveness of the method. The approach worked for all 10 transposons, although they varied in effectiveness based on factors such as temperature and the size of the transposon load.

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

Research also showed that the method could be used with several transposons simultaneously.

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“Instead of just one gene, as is the case with other CRISPR systems such as the more familiar Cas-9 type II system, we can introduce an entire metabolic pathway to incorporate an entirely new set of functions into an organism,” Barrangou said. “In the future, that could mean providing plants with more flexible disease or drought resistance, for example.”

“We are excited by these results and see the potential for applying these newly discovered systems in crop plants to accelerate the development of more resistant, high-yielding varieties,” said Gusui Wu, Global Head of Seed Research for Syngenta Seeds .

Barrangou and Wu add that the work in this study provides a prime example of public-private partnerships driving scientific discovery and shaping tomorrow’s workforce.

The card appears in Nucleic acid research. Funding was provided by Syngenta Seeds. Co-authors of the paper include NC State graduate student Avery Roberts and former NC State Ph.D. student Matthew Nethery.


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