Biology

CRISPR breakthrough uses "hairpin lock" for more precise genetic engineering

CRISPR breakthrough uses "hair...
The new hairpin lock – blue section at the top left – folds back on itself until it finds the target DNA sequence
The new hairpin lock – blue section at the top left – folds back on itself until it finds the target DNA sequence
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The new hairpin lock – blue section at the top left – folds back on itself until it finds the target DNA sequence
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The new hairpin lock – blue section at the top left – folds back on itself until it finds the target DNA sequence

The CRISPR gene-editing system is incredibly accurate, but when there are billions of base pairs of DNA to scroll through, it's not unusual for it to be a little bit off target sometimes. Now, biomedical engineers at Duke University have created an RNA "lock" that can apparently make the system far more precise, and it works with all kinds of CRISPR variations.

In nature, CRISPR is a type of DNA sequence that bacteria use to defend themselves from viruses. After a bug survives an attack, it uses an enzyme to snip out a section of the invader's DNA and store it so it can recognize the same attacker next time. Scientists realized that the mechanism can be co-opted to make precise genetic edits in living organisms, and CRISPR gene-editing was born.

While the technique has proven itself valuable as a potential treatment for a whole host of genetic diseases, it isn't perfect. CRISPR systems use RNA molecules as guides to find the target DNA sequence, but when these guides are scanning through billions of base pairs, they sometimes miss the goal by one or two base pairs. That might not sound like much, but these unintended edits could have some bad side effects.

"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," says Charles Gersbach, corresponding author of the study. "But the solutions proposed thus far cannot be easily translated between different CRISPR systems."

The Duke-developed hairpin lock aims to solve both problems. The common factor to all CRISPR systems is the guide RNA, so the team added an extra 20 nucleotides to the end of it. This new tail is designed to loop back on itself, forming a lock that's hard to break. In fact, the only thing that can open it is the target sequence of DNA, which the RNA tail prefers to bind to. That makes the CRISPR system precise down to a single base pair.

"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," says Dewran Kocak, lead researcher on the study.

The team tested the technique with five different CRISPR variations, and found that the RNA lock boosted the accuracy of edits in cultured human cells by an average of 50 fold. And in one test in particular the method was found to be over 200 times more precise than usual.

The fact that this hairpin lock works on multiple CRISPR systems is crucial, since new versions turn up pretty regularly. Along with the classic Cas9 enzyme, there's Cas12a and Cas12b, which are meant to be safer and more precise; CasX which is smaller so can get into cells easier; and Cas3, which works less like molecular scissors and more like a DNA shredder.

"We're focused on a solution that doesn't add more parts and is general to any kind of CRISPR system," says Kocak. "What's common to all CRISPR systems is the guide RNA, and these short RNAs are much easier to engineer."

The research was published in the journal Nature Biotechnology.

Source: Duke University

1 comment
Biotechz
Very nice! There have been several papers which have previously looked at modifying the stem loops on the sgRNA. However those generally looked at how the guide binds to the Cas9 nuclease differently with these modifications. This "lock" technology instead looks at how these stem loop mods affect binding to the target DNA sequence vs. other sites in the genome where there's partial homology; off-targets. Love this! Anything to reduce off-targets without requiring the re-engineering of each Cas nuclease is a huge win in my book. And they used synthetic sgRNA from Synthego too, which is very cutting-edge.