[ad_1]
DURHAM, NC – Biomedical engineers at Duke University have developed a method to improve the accuracy of CRISPR's genome editing technology by an average of 50 percent. They believe that it can be easily translated into one of the ever-changing formats of editing technology.
The approach adds a short tail to the guide RNA that is used to identify a sequence of DNA to edit. This added tail folds and binds on itself, creating a "lock" that can only be canceled by the targeted DNA sequence.
The study appears online on April 15 in the newspaper Nature Biotechnology.
"CRISPR is usually incredibly accurate, but some examples have shown an untargeted activity, so the field is becoming more and more interested in the specificity," said Charles Gersbach, associate professor of biomedical engineering of the Rooney family to Duke. "But the solutions proposed so far can not be easily translated between different CRISPR systems."
CRISPR / Cas9 is a defense system that bacteria use to target and cleave DNA from invading viruses. While the first version of CRISPR technology designed to work in human cells came from a bacterium called Streptococcus pyogenes, many other species of bacteria carry other versions.
Scientists in the field have spent years researching new CRISPR systems with desirable properties and constantly adding to the CRISPR arsenal. For example, some systems are smaller and better able to integrate with a viral vector for transmission to human cells for gene therapy. But whatever their individual abilities, all have sometimes produced unwanted genetic modifications.
A universal property of CRISPR systems is their use of RNA molecules as guides that refer to the targeted DNA sequence in the genome. Once the guide RNA has found its complementary genetic sequence, the Cas9 enzyme acts as a chisel that cuts steel, thus facilitating genome sequence changes. But since each original head sequence is only 20 nucleotides long and the human genome contains about 3 billion base pairs, there are many things to sort through and CRISPR can sometimes make mistakes with sequences of one or two pairs of bases too short.
One way to improve the accuracy of CRISPR is to require that two Cas9 molecules bind on opposite sides of the same DNA sequence to allow complete cutting. While this approach works, it adds more components to the system, which increases its complexity and makes it harder to deliver.
Another approach has been genetic engineering of the Cas9 protein to make it less energetic, so it is less likely to jump and make mistakes. While this has also yielded promising results, this type of protein engineering is laborious and such efforts are specific to each CRISPR system.
"It seems like a new CRISPR system is being discovered almost every week and that it has some kind of unique property that makes it useful for a specific application," Gersbach said. "Doing a thorough reengineering every time we find a new CRISPR protein to make it more accurate is not a simple solution."
"We are focusing on a solution that does not add additional parts and that applies to any type of CRISPR system," said Dewran Kocak, the PhD student working in the Gersbach lab who led this project. "What is common to all CRISPR systems is the guide RNA, and these short RNAs are much easier to design."
The solution of Gersbach and Kocak is to extend the guide RNA of 20 nucleotides so that it folds on itself and binds to the end of the original guide RNA thus forming a hairpin shape. This creates a kind of lock that is very difficult to move if even a single base pair is incorrect in a desired DNA sequence for a possible cut. But because the guide RNA would prefer to bind to DNA rather than to itself, the correct combination of DNA is still able to break the lock.
"We are able to refine the strength of the lock just enough so that the RNA guide works when it meets the right match," said Kocak.
In this paper, Kocak and Gersbach show that this method can increase by 50 times the precision of sections in human cells on five different CRISPR systems derived from four different bacterial strains. And in one case, this improvement was multiplied by 200.
"It's a pretty simple idea, even though Dewran has conducted several years of research to show that it works as we think," Gersbach said. "It's an elegant and elegant solution to get rid of the non-targeted activity."
In the future, researchers hope to see how many different CRISPR variants could be used with this approach, and complete a detailed characterization of how the locking mechanism works to determine if there are differences between CRISPR variants. And as these experiments were conducted in cultured cells, the researchers are eager to see how this approach could increase the accuracy of CRISPR in a real animal disease model.
###
This work was funded by the Paul G. Allen Borders Group, the National Institutes of Health (DP2OD008586, R01DA036865, R01AR069085, P30AR066527) and the National Science Foundation (DMR-1709527, EFMA-1830957).
QUOTE: "Increase the specificity of CRISPR systems with secondary structures of engineering RNA." D. Dewran Kocak, Eric A. Josephs, Vidit Bhandarkar, Shaunak Adkar S., Jennifer B. Kwon and Charles A. Gersbach. Nature Biotechnology, 2019. DOI: 10.1038 / s41587-019-0095-1
Warning: AAAS and EurekAlert! are not responsible for the accuracy of the news releases published on EurekAlert! contributing institutions or for the use of any information via the EurekAlert system.
[ad_2]
Source link