New CRISPRs expand upon the original’s abilities

Researchers at Duke University and North Carolina State University have discovered a handful of new CRISPR-Cas systems that could add to the capabilities of the already transformational gene editing and DNA manipulation toolbox.

Of the new recruits, one system from bacteria commonly found in dairy cows shows particular promise for human health. Its efficiency is on par with the original and most widely used CRISPR-Cas system, but its small size allows it to be more easily packaged for delivery to human cells. It also can target specific gene sequences that other systems cannot, and human immune systems are unlikely to have been exposed to it.

The results appear online March 14 in the Proceedings of the National Academy of Sciences (PNAS).

CRISPR-Cas9 burst onto the broader scientific scene in 2012 when a team led by Jennifer Doudna showed that it could be modified to target and cut specific segments of DNA. The CRISPR half of the system acts as a genetic homing device, while the Cas9 portion works like a scalpel that cuts where CRISPR tells it to. That work – and most of the subsequent research using CRISPR – is built on a viral defense mechanism from the bacterial species Streptococcus pyogenes. But other bacterial species also have similar defense systems with a wide range of potential abilities and limitations.

“It’s actually remarkable that the first CRISPR-Cas systems researchers used on human cells is still the one that works the best,” said Charlie Gersbach, the John W. Strohbehn Distinguished Professor of Biomedical Engineering at Duke. “We wanted to scour bacteria found in more obscure settings for different CRISPR systems that might have different abilities.”

To do this, Gersbach and his group turned to one of the world’s first and foremost experts on CRISPR, whose lab is just 25 miles down the highway. Seven years before the technology’s Nobel-award-winning paper, Rodolphe Barrangou of NC State characterized CRISPR as a defense system in bacteria used in dairy starter cultures. Since then, his laboratory has focused on exploring the diversity of CRISPR biology for applications ranging from food manufacturing and probiotics to manipulating wood properties by editing tree genomes.

“There is a lot more CRISPR-Cas system diversity in nature than people appreciate, and it can be very useful to mine for diverse effectors with functional potential as molecular machines,” said Barrangou, the Todd R. Klaenhammer Distinguished Professor of Food, Bioprocessing and Nutrition Sciences at NC State. “While some incumbent effectors like Cas9 have shown great potential in the clinic already, we need to expand the CRISPR toolbox for next-generation manipulation of the genome, transcriptome and epigenome.”

Barrangou has developed computational processes for identifying CRISPR-Cas systems within large databases of bacterial genomes. Called “CRISPRdisco,” the program identified more than 1000 different unexplored CRISPR systems, which the researchers whittled down to 50 candidates for Gersbach’s laboratory to generate and further engineer.

Those CRISPR systems were then tested in human cells for their abilities as gene repressors and activators as well as genetic and epigenetic editors. While four systems stood out for their individual successes, one was particularly noteworthy for its versatility across the board. Called SubCas9, the promising CRISPR component was found in Streptococcus uberis, bacteria commonly found in dairy cows that are also used in some human probiotic products.

The researchers are excited about SubCas9 for several reasons. It’s smaller than the traditionally used Cas9 DNA molecular scalpel, meaning it could more easily be loaded into delivery systems that efficienctly transfer cargo to human tissues. It can also target different genetic sequences than its original counterpart. Whereas the most commonly used Cas9 works at genomic targets adjacent to the DNA sequence “GG,” the new system works at sites neighboring “AATA” or “AGTA” patterns.

“GG is a fairly common DNA sequence, but if you really need to target a specific base pair of DNA and there isn’t a GG nearby, then you need an alternative option,” said Gabe Butterfield, the postdoctoral fellow in the Gersbach Lab who co-led the work with Duke biomedical engineering PhD student Dahlia Rohm. “This system can give researchers flexibility for using different Cas9s when they need to be really precise with their target site selection.”

Last but certainly not least, S. uberis is not commonly found in people, in contrast to bacteria species from which the more common Cas9 proteins have been isolated. This means that most people’s immune systems would not recognize SubCas9 from a previous natural exposure if it were to be used in a therapeutic application.

Moving forward, the researchers are working to see if SubCas9 evades preexisting immunity as they expect and are testing incorporating it into a number of cell and gene therapies. They may also dip back into the massive bacterial metagenomic databases that are now available to find more CRISPR systems to investigate.

“Besides potential for therapeutic applications, we also appreciate that bacteria that have adapted to diverse habitats harbor effectors better suited for various kinds of hosts, with much potential for discovery of systems more suited for plants, livestock and environmental applications,” said Barrangou.

“We’ve been collaborating for several years now, and it’s been a really fruitful meshing of Duke’s biomedical abilities and NC State’s agricultural and microbiological expertise,” Gersbach said. “All of our work over the past decade has been made possible by investments from the National Institutes of Health to expand science’s genomic and epigenomic editing toolbox.”

This work was supported by National Institutes of Health (U01AI146356, UM1HG012053, R01MH125236, RM1HG011123), the National Science Foundation (EFMA-1830957), DARPA (HR0011-19-2-0008), the Paul G. Allen Frontiers Group and the Open Philanthropy Project.

CITATION: “Characterization of diverse Cas9 orthologs for genome and epigenome editing.” Gabriel L. Butterfield, Dahlia Rohm, Avery Roberts, Matthew A. Nethery, Anthony J. Rizzo, Daniel J. Morone, Lisa Garnier, Nahid Iglesias, Rodolphe Barrangou, Charles A. Gersbach. PNAS, 2025. DOI: 10.1073/pnas.2417674122