Expanding the CRISPR-based toolbox for extensive genome engineering

Researchers from North Carolina State University describe a variety of CRISPR-Cas systems-based molecular tools to rewrite, rather than just edit, significant portions of an organism’s DNA. These tools are linked to selfish genetic “hitchhikers” called transposons.

The researchers looked at several Type I-F CRISPR-Cas systems and design them to add genetic cargo—up to 10,000 more genetic code letters—to the transposon’s cargo to produce desired changes to a bacterium, in this case, E. coli.

The results broaden the CRISPR toolbox and might have far-reaching ramifications in the manipulation of bacteria and other species at a time when versatile genome editing is required in therapeutics, biotechnology, and more sustainable and efficient agriculture.

Bacteria utilize CRISPR-Cas as adaptive immune systems to combat threats such as viruses. Scientists have used these techniques to remove, cut, and replace certain genetic code sequences in different organisms. The new discovery demonstrates that bigger quantities of genetic information can be transferred or added, possibly improving the functionality of CRISPR.

In nature, transposons have co-opted CRISPR systems to, selfishly, move themselves around an organism’s genome to help themselves survive. We’re in turn co-opting what occurs in nature by integrating with transposons a programmable CRISPR-Cas system that can move around genetic cargo that we design to perform some function.”

Rodolphe Barrangou, Study Corresponding Author and Todd R. Klaenhammer Distinguished Professor, Food, Bioprocessing and Nutrition Sciences, North Carolina State University

Barrangou adds, “Using this method, we showed that we can engineer genomes by moving chunks of DNA up to 10,000 letters. Nature already does this—the bioinformatic data shows examples of up to 100,000 genetic letters moved around by transposon-based CRISPR systems—but now we can control and engineer it by using this system. To complete the hitchhiking analogy, we’re engineering the hitchhiker to bring certain luggage or cargo into the car to deliver some type of payload when the car arrives at its destination.”

The study shows the researchers demonstrating the method’s efficacy in vitro on a lab bench and in vivo in E. coli. The researchers chose 10 different CRISPR-associated transposons to put the strategy to the test. The method worked with all 10 transposons, however, its effectiveness varied depending on factors such as temperature and the quantity of the transposon’s cargo load.

It was exciting to find that all of the systems we tested were functional after reconstructing them into genome editing tools from their native biological forms. We uncovered new features of these systems, but there’s likely many more relevant findings and applications to come as the field moves at a rapid pace.”

Avery Roberts, Study First Author and Graduate Student, North Carolina State University

The study also demonstrated that the approach could be utilized with many transposons at the same time.

Barrangou states, “Instead of just one gene—as is the case with other CRISPR systems like the more familiar Type II Cas-9 system—we can bring in a whole metabolic pathway to incorporate a whole new set of functions to an organism. In the future, that could mean providing more flexible disease resistance or drought resistance to plants, for example.”

We are excited about these findings and see the potential for applying these newly discovered systems in crop plants to accelerate the development of more resilient, higher-yielding varieties.”

Gusui Wu, Global Head, Seeds Research, Syngenta Seeds

According to Barrangou and Wu, the work in this research is an excellent example of public-private partnerships that stimulate scientific discovery and prepare tomorrow’s workforce.