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A better CRISPR? RNA 'hairpins' could improve gene editing

April 17, 2019 By fiercebiotech

One of the biggest worries holding back the burgeoning field of CRISPR gene editing is that the technology can make mistakes, causing off-target effects by cutting the wrong piece of DNA. Engineers at Duke University have devised a solution to the problem they believe will be able to be applied to many forms of CRISPR, even as the technology evolves in the future.

CRISPR consists of a piece of RNA that serves as a guide, and the enzyme Cas9, which cuts the DNA sequence being targeted. The Duke team added a short tail made of up to 20 nucleotides to the RNA, which locks when it reaches the DNA sequence. They described the technique in the journal Nature Biotechnology.

The tail works by folding back and binding to the guide RNA once it reaches its target. This forms a “hairpin” that forces Cas9 to make its cut exactly at that site, according to a statement. In the study, the researchers reported that the method improved the accuracy of gene edits in human cells by fiftyfold on average when tested in five CRISPR systems.

"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," said team member Dewran Kocak, a Ph.D. student at Duke, in the statement.

The quest to improve the safety of CRISPR is vast and wide-ranging. One idea, proposed recently by a team led by University of California, Berkeley CRISPR pioneer Jennifer Doudna, involves using Cas9 variants to keep CRISPR switched off until it reaches its target. Several groups are investigating the potential of using different Cas enzymes to improve the technique, including researchers at the University of Texas at Austin who are studying Cas12a.

The head of the Duke team developing the hairpin method, associate biomedical engineering professor Charles Gersbach, noted that regardless of which enzyme is used or how each system is engineered, all CRISPR methods will still likely rely on guide RNAs.

"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 hairpin tail could potentially be used in any CRISPR system, the researchers believe.

The next step for the Duke team is to test the approach in many more CRISPR variants and to better characterize how the hairpin mechanism is working in those systems. From there, they hope to test the method in an animal model of disease.

by Arlene Weintraub

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