Researchers may have found a way to significantly improve CRISPR/Cas9 gene editing technology.
Biomedical engineers from Duke University have added a short tail to the guide RNA that folds back and binds onto itself to create a lock that can only be undone by the targeted DNA sequence, ultimately improving CRISPR’s accuracy by an average of 50-fold.
“CRISPR is generally incredibly accurate, but there are examples that have shown off-target activity, so there’s been broad interest across the field in increasing specificity,” Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering at Duke, said in a statement. “But the solutions proposed thus far cannot be easily translated between different CRISPR systems.”
The initial version of CRISPR was engineered to work in human cells originated from Streptococcus pyogenes, but scientists have been constantly looking for new CRISPR systems with desirable properties. For example, some systems are smaller and fit better inside a viral vector to deliver gene therapy to human cells.
Despite its position as an emerging gene-editing tool, all CRISPR systems produce unwanted genetic edits from time-to-time because they use RNA molecules as guides that home in on the targeted DNA sequence in the genome. After the guide locates its complementary genetic sequence, the Cas9 enzyme acts as a scissor that cuts the DNA and facilitates changes to the genomic sequence. However, because each homing sequence is only 20 nucleotides long—while the human genome contains about three billion base pairs—gene-editing systems can sometimes make mistakes with sequences that are a few base pairs short of perfection.
In the new version, the researchers extended the guide RNA by as many as 20 nucleotides that enable it to fold back onto itself and bind onto the original guide RNA and form a hairpin shape that is difficult to displace if even a single base pair is incorrect in the sequence. To break the lock, the guide RNA will need to bind to the correct DNA combination rather than itself.
“We’re able to fine-tune the strength of the lock just enough so that the guide RNA still works when it meets its correct match,” Dewran Kocak, the PhD student working in Gersbach’s laboratory who led this project, said in a statement.
In testing, the researchers found that they can improve the accuracy of human cell cuts by an average of 50-fold in five different CRISPR systems derived from four different bacterial strains, with one example yielding a 200-fold improvement.
“It’s a pretty simple idea even though Dewran completed several years’ worth of research to show that it works the way that we think it’s working,” Gersbach said. “It’s a nice, elegant solution for getting rid of off-target activity.”
The researcher’s next plan to test the approach on as many different CRISPR variants as they can and complete an in-depth characterization on exactly how the locking mechanisms works to see if there are differences across different CRISPR variants. They also plan to test the approach using an actual animal model of disease.
There have been attempts to improve CRISPR’s accuracy in the past. Researchers have used two Cas9 molecules to bind onto opposite sides of the same DNA sequence for a complete cut in an approach that is effective but adds more parts to the system, which increases its complexity and makes it harder to deliver.
Another method tried in the past is to genetically engineer the Cas9 protein to make it less energetic, improving its accuracy. However, engineering this protein takes a laborious process that needs to be tailored to each individual CRISPR system.
“It seems like there’s a new CRISPR system being discovered almost every week that has some kind of unique property that makes it useful for a specific application,” Gersbach said. “Doing extensive re-engineering every time we find a new CRISPR protein to make it more accurate is not a straightforward solution.”
The study was published in Nature Biotechnology.