The DNA-editing system CRISPR-Cas9 is revolutionizing how scientists approach genetic problems and diseases. Most researchers use a particular version of the Cas9 protein, derived from the bacterium Streptococcus pyogenes, to alter DNA. But other microbes carry their own versions, which cut genes at different locations and could help researchers design more precise and flexible therapies. For a new study published in Nature Communications, researchers analyzed dozens of Cas9 varieties, uncovering broad diversity in how these “molecular scissors” recognize and snip DNA.
To slice genetic material, Cas9 needs a “guide” RNA molecule that directs the protein to a specific DNA sequence—and that sequence needs to end with a short string of DNA code called a protospacer adjacent motif (PAM). Cas9 can cut DNA only in places where its particular PAM happens to already exist. This stringent requirement limits the locations where researchers can edit genes, says Vilnius University biochemist Virginijus Šikšnys, the study's lead author.
To broaden their options, Šikšnys says, the biologists searched through “thousands of sequences of Cas9 proteins in databases” and settled on 79 candidates from different bacteria. They built each Cas9 in a liquid that simulates the cellular interior, and then they added snippets of DNA with random PAM codes to each mixture.
Most Cas9 proteins ended up recognizing a unique PAM sequence—a feature that could let bioengineers cut DNA in more places, says Patrick Pausch, a CRISPR researcher at the University of California, Berkeley, who was not involved in the study. The proteins' varying sizes (one, from a Yellowstone geyser microbe, was 30 percent smaller than standard) could also fit different delivery systems, Šikšnys says.
And having more Cas9 options could potentially help researchers circumvent immune responses to some gene-editing therapies. A prior study found that in 125 blood donors, 58 percent had antibodies against Cas9 derived from S. pyogenes. Cas9 proteins from more innocuous bacteria might be less likely to trigger a bad reaction.
“I think that they are providing a real service to the community by characterizing such a huge range of different proteins,” says Iowa State University biochemist Dipali Sashital, who was not involved in the research. Sashital says there is potential for new tools but notes that the work is foundational; the team has not yet shown that the proteins will work in real-world gene editing.
The researchers next plan to test some of the proteins in living organisms. Regardless of their performance, this large collection illustrates the remarkable diversity of these DNA cutters, Šikšnys says: “It's like having multiple scissors instead of a single pair.”