In a new study, North Carolina State University researchers have described a set of molecular tools to rewrite—not just modify—large pieces of an organism’s DNA, based on CRISPR-Cas systems linked to selfish genetic “wankers” called transposons.
Researchers are investigating and engineering various type IF CRISPR-Cas systems to add a genetic cargo — up to 10,000 additional genetic code letters — to the transposon cargo to make the desired changes to bacteria — in this case, coli.
The findings expand CRISPR’s toolbox and could have major implications for manipulating bacteria and other organisms at a time when flexible genome modification is needed for more sustainable and efficient therapies, biotechnology, and agriculture.
Bacteria use CRISPR-Cas technology as an adaptive immune system to fight off attacks from enemies such as viruses. These systems have been adapted by scientists to remove, cut, and replace specific genetic code sequences in a variety of organisms. The new discovery shows that larger amounts of genetic code can be moved or added, which could increase CRISPR’s functionality.
“In nature, transposons have chosen CRISPR systems, selfishly, to move themselves around the genome of an organism to help themselves survive. We, in turn, choose what happens in nature by integrating transposons with a programmable CRISPR-Cas system that can help themselves survive,” said Rudolph Barangu. Todd R. Kleinhammer Distinguished Professor of Food, Bioprocessing, and Nutritional Sciences at NC State and corresponding author of a paper describing the research: “Moving around genetic cargo that we design to perform certain functions.”
“Using this method, we have shown that we can engineer the genome by transferring pieces of DNA of up to 10,000 letters,” Barango said. “Nature already does this — bioinformatics data shows examples of up to 100,000 genetic letters transferred by transposon-based CRISPR systems — but we can now control and engineer it with this system.”
“To complete the hitchhiking analogy, we engineer the traveler to bring some baggage or cargo into the vehicle to deliver some kind of payload when the vehicle arrives at its destination.”
The study showed that the researchers demonstrated the effectiveness of this method in vitro on the laboratory bench and in vivo coli. The researchers selected 10 different CRISPR-related transposons to test the method’s effectiveness. The approach worked for all 10 transposons, although their effectiveness varied based on factors such as temperature and the size of the cargo payload being transported.
“It was exciting to find that all of the systems we tested were functional after being reconstructed into genome editing tools from their original biological forms,” said Avery Roberts, a 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 results and applications to come as the field moves at a rapid pace.”
The research also showed that the method can be used with different transposons at the same time.
“Instead of just one 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 integrate a whole new set of functions for the organism,” Barrangou said. “In the future, that could mean providing more resilient disease or drought resistance to plants, for example.”
“We are excited about these results and see the potential to apply these newly discovered systems in crop plants to accelerate the development of more resilient, high-yielding varieties,” said Josui Wu, global head of seed research at Syngenta Seeds.
Barango Wu adds that the work on this study provides a great example of public-private partnerships driving scientific discovery and training the workforce of the future.
The sheet appears in Nucleic acid research. Funding was provided by Syngenta Seeds. Co-authors on the research include NC State graduate student Avery Roberts and former NC State Ph.D. Student Matthew Nyeteri.