The & # 39; forks & # 39; Engineering Increase CRISPR Accuracy



The & # 39; forks & # 39; Engineering Increase CRISPR Accuracy

Representation of an artist of the CRISPR / Cas9 genetic sequence with the "hairpin" lock added to the left side of the system, which increases its accuracy by an average of 50 times. Credit: Ella Maru

Biomedical engineers at Duke University have developed a method to improve the accuracy of CRISPR genome editing technology by an average of 50 times. They believe that it can be easily translated into any of the continuously expanding formats of the editing technology.

The approach adds a small tail to the guiding RNA, which is used to identify a DNA sequence to edit. This aggregate tail folds back and binds itself, creating a "lock" that can only be undone by the target DNA sequence.

The study appears online on April 15 in the magazine. Biotechnology of nature.

"CRISPR is generally incredibly accurate, but there are examples that have shown off-target activity, so there has been a lot of interest in the field because of the increasing specificity," said Charles Gersbach, badociate professor of Biomedical Engineering for the Rooney family at 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 attack and cleave DNA from invading viruses. While the first version of CRISPR technology designed to work on human cells originated from a bacterium called Streptococcus pyogenes, many more species of bacteria have other versions.

Scientists in the field have spent years searching for new CRISPR systems with desirable properties and are constantly adding to the CRISPR arsenal. For example, some systems are smaller and more capable of fitting into a viral vector to deliver to human cells for gene therapy. But regardless of their individual abilities, they have all produced unwanted genetic issues at times.

A universal property of CRISPR systems is their use of RNA molecules as guides that focus on the targeted DNA sequence in the genome. Once a guiding RNA finds its complementary genetic sequence, the Cas9 enzyme acts like the scissors that make the cut in the DNA, facilitating the changes in the genome sequence. But because each start sequence has only 20 nucleotides and the human genome contains approximately three billion base pairs, there is much to clbadify and the CRISPR can sometimes make errors with sequences of one or two base pairs below of perfection.

One way to improve the accuracy of CRISPR is to require that two Cas9 molecules bind to opposite sides of the same DNA sequence so that a complete cut is made. While this approach works, it adds more parts to the system, which increases its complexity and makes it more difficult to deliver.

The & # 39; forks & # 39; Engineering Increase CRISPR Accuracy

Charles Gersbach, Associate Professor of Biomedical Engineering for the Rooney Family at Duke University. Credit: Les Todd

Another approach has been to genetically engineer the Cas9 protein to make it less energetic, making it less likely to jump the gun and make an error. While this has also shown promising results, this type of protein engineering is laborious and such efforts are specific to each CRISPR system.

"It seems that a new CRISPR system is being discovered almost every week that has some kind of unique property that makes it useful for a specific application," Gersbach said. "Extensive reengineering every time we find a new CRISPR protein to make it more accurate is not an easy solution."

"We are focused on a solution that does not add more parts and is general for any type of CRISPR system," said Dewran Kocak, Ph.D. Student working in the Gersbach laboratory that directed this project. "What is common to all CRISPR systems is RNA guidance, and these short RNAs are much easier to design."

The solution of Gersbach and Kocak is to extend the guide RNA up to 20 nucleotides so that it folds back on itself and attaches to the end of the original guide RNA, forming a hairpin shape. This creates a kind of blockage that is very difficult to move if even a single base pair is incorrect in a DNA sequence that is badyzed for a possible cut. But because the guiding RNA would prefer to bind to DNA instead of to itself, the correct combination of DNA can still break the blockage.

"We can adjust the strength of the lock enough so that the guide RNA still works when it meets its correct match," Kocak said.

In the paper, Kocak and Gersbach show that this method can increase the accuracy of cuts made in human cells by an average of 50 times in five different CRISPR systems derived from four different bacterial strains. And in one case that improvement was raised more than 200 times.

"It's a pretty simple idea even though Dewran completed several years of research to show that it works the way we think it works," Gersbach said. "It's a nice and elegant solution to get rid of off-target activity."

In the future, researchers hope to see how many different CRISPR variants this approach might work with, as well as complete a deep characterization of exactly how the blocking mechanism works to see if there are differences between the CRISPR variants. And because these experiments were carried out on cultured cells, researchers are eager to see how well this approach could increase CRISPR accuracy within an actual animal model of disease.


New strategy improves the efficiency of the CRISPR-Cas9 genome edition


More information:
Increase the specificity of CRISPR systems with secondary RNA structures designed, Biotechnology of nature (2019). DOI: 10.1038 / s41587-019-0095-1, https://www.nature.com/articles/s41587-019-0095-1

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Duke University


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The & # 39; forks & # 39; of engineering increase CRISPR accuracy (2019, April 15)
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